Cfp1 integrates both CpG content and gene activity for accurate H3K4me3 deposition in embryonic stem cells

Trimethylation of histone H3 Lys 4 (H3K4me3) is a mark of active and poised promoters. The Set1 complex is responsible for most somatic H3K4me3 and contains the conserved subunit CxxC finger protein 1 (Cfp1), which binds to unmethylated CpGs and links H3K4me3 with CpG islands (CGIs). Here we report that Cfp1 plays unanticipated roles in organizing genome-wide H3K4me3 in embryonic stem cells. Cfp1 deficiency caused two contrasting phenotypes: drastic loss of H3K4me3 at expressed CGI-associated genes, with minimal consequences for transcription, and creation of “ectopic” H3K4me3 peaks at numerous regulatory regions. DNA binding by Cfp1 was dispensable for targeting H3K4me3 to active genes but was required to prevent ectopic H3K4me3 peaks. The presence of ectopic peaks at enhancers often coincided with increased expression of nearby genes. This suggests that CpG targeting prevents “leakage” of H3K4me3 to inappropriate chromatin compartments. Our results demonstrate that Cfp1 is a specificity factor that integrates multiple signals, including promoter CpG content and gene activity, to regulate genome-wide patterns of H3K4me3

A Temporal Threshold for Formaldehyde Crosslinking and Fixation

Formaldehyde 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.Using 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.Our findings for the first time establish a minimum temporal limitation to crosslink chemistry that has implications for many fields of research

Inverse relationship between MeCP2 residence time on heterochromatin and the ability to crosslink this interaction by formaldehyde.

<p>A) In vivo residence times on heterochromatin of MeCP2 mutants were determined by FRAP. Mutants with a residence time above 5 seconds were 100% localized in fixed cells, whereas those with shorter residence times localized partially or not at all. B) Immunoprecipitation of formaldehyde-crosslinked MeCP2 is inefficient when the residence time is below 4 seconds. The mutants in order from left to right in panels A and B are: R168X, R106W, R111G, D97E, T158M, L100V, R133C and wildtype. Error bars on both axes correspond to ±standard deviation.</p

Major differences in nuclear localisation of MeCP2-GFP in living versus paraformaldehyde-fixed mouse fibroblasts.

<p>All MeCP2 mutants (labelled left) localized to nuclear foci corresponding to peri-centromeric heterochromatin in living cells, but many showed diffuse nuclear staining in the same cells after paraformaldehyde-fixation.</p