Molecular basis of pioneer transcription factor function revealed by Cryo-EM

• Funding – self-funded/externally sponsored applicants  (PhD Fees can be found here)
• Applications are accepted year round
• Standard Entry dates – January and September
• Applicants are expected to have a degree (equivalent of Honours or Masters) in a relevant discipline.

Research question

The life of mammals starts with fertilization of an egg, which subsequently undergoes multiple rounds of cell division and develops into a complete organism. During mammalian pre-implantation, transcription factors (TFs) play a crucial role in the first lineage segregation into the inner cell mass and trophectoderm (TE), which give rise to the embryo proper and extraembryonic tissue (e.g. placenta), respectively. Among TFs, specialized TFs known as pioneer TFs, play key roles in cell differentiation and reprogramming. Pioneer TFs have unique abilities that bind nucleosomal DNA and modulate epigenetic and chromatin states by facilitating the recruitment of chromatin remodelers and histone modifiers. CDX2 and GATA3 are key pioneer TFs that commit TE cell lineage in mice. However, how these pioneer TFs drive TE gene regulatory networks at the chromatin level during the first lineage segregation remains poorly understood. Our research will establish the fundamental mechanisms underpinning TE lineage commitment, offering a new perspective on gene regulation with far-reaching implications for chromatin biology.

Kobayashi lab uses a multidisciplinary approach to address this question using state-of-the-art methods, including low-input genomics, proteomics, biochemistry, and structural biology. We seek to unravel fundamental principles of gene regulation and epigenetic reprogramming in mammalian development and gain insights into defects in the process that affect fertility.

The project

This PhD project aims to uncover the molecular basis by which pioneer TFs CDX2 and GATA3 engage with nucleosomes and associated co-factors to function as transcriptional activators and repressors. The student will purify recombinant proteins and reconstitute defined complexes with nucleosomes in vitro. Using biochemical and biophysical approaches, the student will investigate how pioneer TFs engage with nucleosomes together with specific co-factors and how these interactions alter chromatin structure. The reconstituted complexes will be further visualized using cryo-electron microscopy (cryo-EM) to determine their molecular basis. If successful, the project also provide training in embryology to investigate how specific amino acid(s) impact on developmental outcomes and gene regulation in vivo. 

What you will gain

• Expertise in recombinant protein purification and in vitro nucleosome reconstitution
• Experience in designing and analysis for biochemical and biophysical experiments
• Training in structural biology using cryo-EM
• Skills for single-particle analysis using software such as RELION and CryoSPARC
• Training in mouse embryology techniques

Related publications:

Kobayashi W, Michael AK, Ruangroengkulrith S, Kümmecke M, Tachibana K. 

Protocol for integrative analysis of transcription factor-nucleosome interactions using SeEN-seq and cryo-EM structure determination. 

STAR Protoc. 2025, 7:104295. 

Kobayashi W, Sappler H. A, Bollschweiler D, Kümmecke M, Basquin J, Arslantas E, Ruangroengkulrith S, Hornberger R, Duderstadt K, Tachibana K. 

Nucleosome-bound NR5A2 structure reveals pioneer factor mechanism by minor groove anchor competition. 

Nat Struct Mol Biol. 2024, 31:757-766. 

Gassler J*, Kobayashi W*, Gáspár I*, Ruangroengkulrith S*, Mohanan A, Gómez Hernández L, Kravchenko P, Kümmecke M, Lalic A, Rifel N, Ashburn RJ, Zaczek M, Vallot A, Cuenca Rico L, Ladstätter S, Tachibana K. 

Zygotic genome activation by the totipotency pioneer factor Nr5a2. 

Science. 2022, 378:1305-1315.

Our research community thrives on the diversity of students and staff which helps to make the University of Dundee a UK university of choice for postgraduate research.  We welcome applications from all talented individuals and are committed to widening access to those who have the ability and potential to benefit from higher education.