Background and Aims. The Ikaros family of sequence-specific DNA binding proteins is central to haematopoiesis, and the founding member IKZF1 is recurrently mutated in human progenitor B cell acute lymphoblastoid leukaemia 1 (B-ALL). How Ikaros regulates transcription has been of long-standing interest 2-4. Time-resolved analysis shows that Ikaros binding drives rapid transcriptional repression of developmentally regulated genes in B cell progenitors 3,4.
Emerging evidence indicates that the transcription of cell type-specific genes is highly organised within the nucleus. RNA polymerase, Mediator, and other components of the transcriptional machinery form nuclear condensates with features of liquid-liquid phase separation 5,6 (LLPS). Cell type-specific enhancers are thought to have a critical role in this emerging principle of transcriptional control 6. Biochemical data show that Ikaros inactivates a subset of B cell-specific enhancers within minutes of entering the nucleus (Tianyi Zhang with Matthias Merkenschlager, LMS).
Ikaros forms obligate dimers of ~100kD as well as higher-order complexes in the megadalton range by mechanisms that involve protein-protein and protein-DNA interactions7. Biochemical and functional data (Tianyi Zhang, LMS), combined with bioinformatics and machine learning approaches show that the gene regulatory impact of Ikaros scales with the density of target sites (with Yi-Fang Wang and Nehir Nebioglu, LMS, and Maxwell Libbrecht, SFU), indicating that Ikaros multimerisation contributes to gene regulation.
In addition to acting on highly transcribed genes in transcriptionally active nuclear domains, Ikaros also forms prominent foci at pericentromeric regions that are enriched in the heterochromatin binding protein HP1a (ref. 2,8). Interestingly, HP1a has been shown to drive the separation of inactive chromatin by a process that resembles LLPS (ref. 9,10). Moreover, the stable silencing of Ikaros target genes is accompanied by their repositioning from transcriptionally active regions in the nucleus to pericentromeric heterochromatin domains 2,3,8.
These findings raise the question how Ikaros interacts with transcriptional active as well as repressive nuclear compartments. To address this question we propose a high resolution approach that combines light and electron microscopy.
Approach. Interactions of wild-type and mutant Ikaros complexes with specific DNA targets will be explored by Immuno-FISH/SIM (with Drs Tianyi Zhang and Karen Brown, LMS). The localisation of Ikaros complexes relative to active and repressive pericentromeric heterochromatin domains will be explored by SRM of tagged Ikaros and Mediator or HP1a, respectively. These experiments will use HALO-tagged Ikaros and SNAP-tagged Mediator or HP1a (ref. 11) in a new collaboration with Prof Marisa Martin-Fernandez and Dr Lin Wang (UKRI Science and Technology Facilities Council, RFI), and supported by Prof David Rueda's Single molecule Imaging Group (ICL/LMS).
Correlative light and cryo-electron microscopy (cryoCLEM) is an emerging technology for the structural analysis of protein complexes in their native subcellular localisation12. cryoCLEM uses light microscopy to identify regions of interest and employs electron microscopy to reveal structural details within these regions. We will utilise the power of cryoCLEM to visualise Ikaros-GFP transcriptionally active and repressive compartments, which will be identified using Mediator-RFP and HP1a-RFP fusion proteins, respectively. Localisation by light microscopy will be followed first by serial cryoFIB/SEM volume imaging to explore overall structural features of the whole nucleus, and then by high resolution cryo-electron tomography of cryoFIB milled thin cell lamella focusing on higher order Ikaros complexes at transcriptionally active or repressive nuclear compartments
The cryoCLEM experiments described here are high risk/high gain. In the event that further technical developments are needed before this emerging technology can be fully applied to our research question, we will harness the biochemistry skills of Dr Tianyi Zhang (LMS) and cryoEM expertise of Prof Zhang's team (eBIC/RFI) to perform cryoEM studies of affinity purified and size-separated native Ikaros complexes7, and of recombinant Ikaros assembled on DNA templates that differ in the number and the arrangement of Ikaros binding sites.
Why this project is important. How enhancer activity is regulated is a critical question for cell lineage commitment and differentiation. Ikaros-mediated control of enhancer activity is an important paradigm because Ikaros is central for normal haematopoiesis and human B-ALL.
Why this project will make a great PhD studentship. This project offers interdisciplinary training based on a tractable cell biological system, which is amenable to both established approaches (biochemical analysis, SRM, computational biology) and emerging approaches (CLEM). Both eBIC/RFI and the LMS bring strong expertise in their respective fields. The student will enjoy an exceptional support network comprising expertise in genetics, cell biology, biochemistry, bioinformatics, and machine learning, and will apply cutting edge imaging to break new ground in gene regulation.
To apply for this programme please visit the LMS website where the application form is available for download - https://lms.mrc.ac.uk/study-here/phd-studentships/lms-3-5yr-studentships/
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