Epigenetics of eu- and heterochromatin in inverted and conventional nuclei from mouse retina.

To improve light propagation through the retina, the rod nuclei of nocturnal mammals are uniquely changed compared to the nuclei of other cells. In particular, the main classes of chromatin are segregated in them and form regular concentric shells in order; inverted in comparison to conventional nuclei. A broad study of the epigenetic landscape of the inverted and conventional mouse retinal nuclei indicated several differences between them and several features of general interest for the organization of the mammalian nuclei. In difference to nuclei with conventional architecture, the packing density of pericentromeric satellites and LINE-rich chromatin is similar in inverted rod nuclei; euchromatin has a lower packing density in both cases. A high global chromatin condensation in rod nuclei minimizes the structural difference between active and inactive X chromosome homologues. DNA methylation is observed primarily in the chromocenter, Dnmt1 is primarily associated with the euchromatic shell. Heterochromatin proteins HP1-alpha and HP1-beta localize in heterochromatic shells, whereas HP1-gamma is associated with euchromatin. For most of the 25 studied histone modifications, we observed predominant colocalization with a certain main chromatin class. Both inversions in rod nuclei and maintenance of peripheral heterochromatin in conventional nuclei are not affected by a loss or depletion of the major silencing core histone modifications in respective knock-out mice, but for different reasons. Maintenance of peripheral heterochromatin appears to be ensured by redundancy both at the level of enzymes setting the epigenetic code (writers) and the code itself, whereas inversion in rods rely on the absence of the peripheral heterochromatin tethers (absence of code readers).

Reliable detection of epigenetic histone marks and nuclear proteins in tissue cryosections.

Nuclear processes in real tissues often are significantly different from those in cultured cells. However, immunostaining on tissue sections needs long fixation which masks antigens and, respectively, antigen retrieval which restores antigen accessibility. These treatments affect the immunostaining results and complicate their interpretation. The problem is especially significant for nuclear antigens which often are very sensitive to both fixation and antigen retrieval. We targeted this problem by a study of several histone modifications and nuclear proteins in tissue sections of mouse retina which contains cells with both conventional and unique inverted nuclei. In the latter, the main chromatin classes form separate concentric shells which simplifies evaluation of the signal distribution. We show that as a rule, longer fixation demands longer antigen retrieval time. Nevertheless, antigens are remarkably diverse in this respect and need individual adjustment. We suggest a robust procedure for immunostaining on sections, that is, a method that allows controlling the differences in immunostaining caused by differences in fixation time and antigen retrieval duration, so that immunostaining protocol can be quickly optimized.