Fig. 5

Specific targeting of histone acetyltransferase leads to hyperacetylation and increase in DNA replication duration of constitutive heterochromatin. a Schematic representation of the targeting approach in a Microtus cabrerae cell. HBO1, a histone acetyltransferase, was tagged to GFP, and HP1-beta was tagged to GBP, a GFP-binding protein. Upon co-expression of both and because of their strong interaction, HBO1 is specifically targeted to the H3K9me3 decorated X chromosome. The experimental setup (middle box) implied the transient transfection of two plasmids: GFP-HBO1 and GBP-HP1-beta, followed by an incubation time of 24 h. The functionality of the targeting was validated by antibody detection of H3K9me3 a marker of constitutive heterochromatin, resulting in a strong colocalization of DAPI-stained DNA in blue, GFP-HBO1 and GBP-HP1-beta in green and H3K9me3 in red. The merge shows an overlay of all three channels. Scale bar = 5 µm. b Untargeted and targeted cells were analyzed with a user-independent analysis to measure H3K9me3 and acetylation level at the X chromosome. Bar graphs indicate the ratio of the mean levels, where grayish bars represent the normalized control and colored bars the respective targeted sample. Statistical significance was tested using the t test, comparing untargeted and targeted cells. Error bars demonstrate 95 Cl. ***P < 0.001. c Untargeted and targeted cells were analyzed with a user-independent analysis to measure H3K9me3 and acetylation levels in the whole nucleus excluding the H3K9me3 decorated X chromosome. Bar graphs indicate the ratio of the mean levels, where grayish bars represent the normalized control and colored bars the respective targeted sample. Statistical significance was tested using the t test, comparing untargeted and targeted cells. Error bars demonstrate 95 Cl. d The duration of X chromosome replication was estimated out of live-cell imaging data. Error bars demonstrate standard deviation. Statistical significance was tested using the t test, comparing the duration of replication of X* and X in untargeted and targeted samples. As a negative control, a catalytically dead HBO point mutant (G485A) was used. ***P < 0.001