Abstract: FR-PO1002
ChromA-ExM: A New Application of Expansion Microscopy for Optical Imaging of Chromatin Architecture in Renal Tubular Epithelial Cells with Nanoscale Resolution
Session Information
- Pathology and Lab Medicine: Basic
November 08, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Pathology and Lab Medicine
- 1601 Pathology and Lab Medicine: Basic
Authors
- Soliman, Kirolous, Brigham and Women's Hospital, Boston, Massachusetts, United States
- Santos, Nicole D., Brigham and Women's Hospital, Boston, Massachusetts, United States
- Sobral reyes, Maria Fernanda, Brigham and Women's Hospital, Boston, Massachusetts, United States
- Hernández ramos, Joel Orlando, Brigham and Women's Hospital, Boston, Massachusetts, United States
- Lemos, Dario R., Brigham and Women's Hospital, Boston, Massachusetts, United States
Group or Team Name
- Laboratory for Organ Regeneration and Tissue Engineering
Background
In the eukaryotic cell nucleus, chromatin is physically organized into euchromatin and heterochromatin domains. Those domains regulate transcriptional accessibility to DNA, ultimately determining cell phenotype and function. Currently, high-resolution visualization of chromatin domain architecture can be achieved only with either electron microscopy or super-resolution microscopy. Both techniques have practical limitations, a major one being that they are not easily accessible to most laboratories. Here we introduced a modification to the original Expansion Microscopy (ExM) protocol developed at MIT that allows nanoscale resolution for visualization of high-order chromatin structures using regular confocal microscopes. The new procedure is called Chromatin Architecture Expansion Microscopy (ChromA-ExM)
Methods
Using conventional immunofluorescence techniques we labeled histone decorations in either cell or tissue specimens. The immunostained tissue specimens are subsequently embedded in a swellable polymer network, and digested with protease K. The combination of both treatments allows for ~4.5 isotropic expansion of the sample, which constitutes a magnification factor multiplied by the magnification of the microscope objective used. In ChromA-ExM we introduced steps of selective enzymatic digestion with DNAses allowing further expansion of chromatin domains
Results
Analysis of H3K9me3 chromatin domains in kidney PTECs indicated that, compared to regular ExM, the ChromA-ExM protocol results in increased resolution for visualization of high-order chromatin domains, including chromocenters associated with active cell phenotype, and senescence associated heterochromatin foci. Further, we can interrogate spatial interactions between H3K9me3+ (silent chromatin) and H3K4me3+ (open chromatin) domains in kidney tubular epithelial cells with exquisite detail, to detect architectural chromatin arrangements associated with kidney disease
Conclusion
ChromA-ExM is a new tool to study chromatin spatial configuration and chromatin status. The technique is useful for ultrastructural analysis of chromatin changes in PTEC senescence and tubular pathologies
Funding
- Other NIH Support