Abstract
BackgroundFormaldehyde crosslinking is in widespread use as a biological fixative for microscopy and molecular biology. An assumption behind its use is that most biologically meaningful interactions are preserved by crosslinking, but the minimum length of time required for an interaction to become fixed has not been determined.MethodologyUsing a unique series of mutations in the DNA binding protein MeCP2, we show that in vivo interactions lasting less than 5 seconds are invisible in the microscope after formaldehyde fixation, though they are obvious in live cells. The stark contrast between live cell and fixed cell images illustrates hitherto unsuspected limitations to the fixation process. We show that chromatin immunoprecipitation, a technique in widespread use that depends on formaldehyde crosslinking, also fails to capture these transient interactions.Conclusions/SignificanceOur findings for the first time establish a minimum temporal limitation to crosslink chemistry that has implications for many fields of research.
Highlights
Chemical crosslinking with formaldehyde and related reagents is widely used to fix sub-cellular structures for microscopy and to immobilise protein-DNA contacts for chromatin immunoprecipitation (ChIP [1,2,3,4])
We have found that this assumption becomes invalid when intermolecular contacts are short-lived. This limitation to formaldehyde crosslinking became apparent via our studies of the methylated DNA binding protein MeCP2, which associates in a DNA methylation-dependent manner with heterochromatic foci in mouse cell nuclei [5,6]
A series of mutants of the MeCP2 DNA binding domain fail to localize to heterochromatin in fixed cells, but localize indistinguishably from wildtype protein when living cells are examined by fluorescence microscopy
Summary
Chemical crosslinking with formaldehyde and related reagents is widely used to fix sub-cellular structures for microscopy and to immobilise protein-DNA contacts for chromatin immunoprecipitation (ChIP [1,2,3,4]). Exposure of living cells to formaldehyde results in covalent linkage with exposed amino and imino groups (notably in lysine and arginine sidechains). This forms a Schiff’s base that can participate in a second linkage, creating methylene bridges between amino acids that were in close proximity (,2 A ) in the native protein. We have found that this assumption becomes invalid when intermolecular contacts are short-lived This limitation to formaldehyde crosslinking became apparent via our studies of the methylated DNA binding protein MeCP2, which associates in a DNA methylation-dependent manner with heterochromatic foci in mouse cell nuclei [5,6]. An assumption behind its use is that most biologically meaningful interactions are preserved by crosslinking, but the minimum length of time required for an interaction to become fixed has not been determined
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