Extended Data Fig. 5: Mo-2 regions accumulate heterochromatin factors.

a, Costaining of ANKRD31 or mo-2 with the indicated proteins and histone marks known to localize at the pericentromeric heterochromatin (mouse major satellite), in zygotene spermatocytes (left) and pre-leptotene spermatocytes (right). Each of the heterochromatin factors shows a locally enriched signal coincident with mo-2 regions (arrowheads), in addition to broader staining of other sub-nuclear regions. The CHD3/4 antibody recognizes both proteins⁴⁹. The colocalization of ANKRD31 blobs with heterochromatin blobs was observed in all zygotene spermatocytes analysed (n > 20) in at least three mice for each antibody (left) and in one mouse for pre-leptotene spermatocytes (n > 10) for each antibody (right). Scale bars, 2 μm. b, CHD3/4, ATRX, HP1β, H4K20me3, H3K9me3 and macroH2A1.2 are not detectably enriched at mo-2 regions in spermatogonia (small, DMRT1-positive cells). These factors may be present at mo-2 regions in these cells, but do not appear to accumulate to elevated levels. The absence of colocalization between mo-2 FISH signals and heterochromatin factors was noted in all spermatogonia analysed (n > 30) from one mouse. Scale bars, 2 μm. c, Heterochromatin factors can be detected in the PAR up to late pachynema. Each of the assayed proteins and histone marks showed staining on the autosomal and X-specific pericentromeric heterochromatin, the sex body and euchromatin—albeit with variations between sites in the timing and level of accumulation. Notably, however, they also showed enriched staining at all mo-2 regions up to early or mid-pachynema, as shown for H4K20me3 (top). By mid-to-late pachynema, as shown here for H3K9me3, the signal persisted in the PAR but was usually barely detectable at mo-2 regions of chromosome 9 or chromosome 13. This observation indicates that—at least for the PAR—the heterochromatin factors can continue to be enriched on mo-2 chromatin after RMMAI proteins have dissociated. These results substantially extend previous observations about CHD3/4 colocalizing with PAR FISH signals; H4K20me3 being localized in the PAR and at the ends of other chromosomes; and detection of H3K9me3, HP1β and macroH2A1.2 in the PAR in late pachynema^49,50,51,52. The colocalization between major satellite (maj sat) and H4K20me3 and H3K9me3 was observed in all spermatocytes analysed (n > 20) in one mouse. The colocalization between H4K20me3 and mo-2 FISH signals was observed in all spermatocytes analysed (n > 60) from the pre-leptotene to mid-pachytene stage in more than three mice. Scale bars, 2 μm. d, Enrichment of the heterochromatin factors is independent of SPO11. Representative images of Y chromosomes from a Spo11^–/– mouse are shown. The colocalization between PAR mo-2 FISH signals and heterochromatin factors was observed in all Spo11^−/− spermatocytes analysed (n > 30) in more than three mice for CHD3/4 and at least one mouse each for ATRX, HP1β, HP1γ, macroH2A1.2, H3K9me3 and H4K20me3. Scale bar, 1 μm.