The Role of architectural proteins on interphase chromatin topology and transcriptional regulation

Ezequiel J. Miron S.. James Rhodes. Quentin Alle.

Chromatin conformation studies on the cohesin complex and the CCCTC binding factor (CTCF) have shown them to be key regulators of Mb-scale looping and consequent Mb-sized topologically associated domains (TADs) on the linear genome. The way this linear architecture relates to the 3D topology of interphase chromatin in mammalian cells as imaged by super-resolution microscopy is still to be elucidated. Through the use of a dedicated image analysis workflow we are quantifying the re-distribution of epigenetic marks (implicated in transcriptionally active and silent genes as well as inter-genic regions) as a read out for comparing physiological state to that caused TEV protease knockout and RNAi knockdowns of cohesin and CTCF.



Novel CRISPR modality for versatile super-resolution chromatin imaging

Ezequiel J. Miron S.. Andrew Bassett.

Developing an alternative approach to circumvent previous limitations of CRISPR-based imaging for single loci and live cell super resolution microscopy. Here we demonstrate the use of multiple in vitro-labelled single guide (sg) RNAs, whilst maintaining the specificity and activity of the Cas9 nuclease. 

The ability to increase the signal per molecule becomes critical when considering single-copy gene loci cannot compensate for low signal-to-noise ratios through a high local concentration of tandem repeat target sequences. sgRNAs with different labels could also be combined in multi-channel structured illumination microscopy (SIM) to address questions on chromatin organisation and dynamics with in vivo super-resolution. 

This technique could be extended to simultaneously image chromatin and trigger changes to its structure and function, through further Cas9 protein fusions and recruitment of nuclear machinery components. We envision a non-denaturing alternative to fluorescence in situ hybridisation (FISH) approaches that is easily adapted to new target sites and is amenable to live-cell studies.

Chromatin segmentation



ABOUT

Ezequiel graduated in Biochemistry from Imperial College London where he developed an interest in gene regulation and transcription from his work with archaeal RNA Polymerase. His PhD focuses on the role of key chromatin architectural proteins on interphase chromatin topology as characterised by the three-dimensional re-distributions of epigenetic markers by super-resolution microscopy,

Native of Argentina, he enjoys good food, good music and good company (not necessarily in that order).

Eze.Miron@bioch.ox.ac.uk