Centromere inactivation on a neo-Y fusion chromosome in threespine stickleback fish

Abstract

Having one and only one centromere per chromosome is essential for proper chromosome segregation during both mitosis and meiosis. Chromosomes containing two centromeres are known as dicentric and often mis-segregate during cell division, resulting in aneuploidy or chromosome breakage. Dicentric chromosome can be stabilized by centromere inactivation, a process which reestablishes monocentric chromosomes. However, little is known about this process in naturally occurring dicentric chromosomes. Using a combination of fluorescence in situ hybridization (FISH) and immunofluorescence combined with FISH (IF-FISH) on metaphase chromosome spreads, we demonstrate that centromere inactivation has evolved on a neo-Y chromosome fusion in the Japan Sea threespine stickleback fish (Gasterosteus nipponicus). We found that the centromere derived from the ancestral Y chromosome has been inactivated. Our data further suggest that there have been genetic changes to this centromere in the two million years since the formation of the neo-Y chromosome, but it remains unclear whether these genetic changes are a cause or consequence of centromere inactivation.

Keywords: CENP-A; Centromere inactivation; ChIP-seq; Dicentric chromosome fusion; Gasterosteus aculeatus; Gasterosteus nipponicus.

Fig. 1

The Japan Sea neo-Y is a fusion between two complete chromosomes. FISH with the ancestral X and Y chromosome BACs 188J19 (purple) and101E08 (green) and the chromosome 9 BAC 44L12 (purple) on a Japan Sea male metaphase spread shows (a) three chromosomes with BAC hybridization each highlighted by square boxes. Higher magnification of the boxed regions in panel (a) shows the neo-Y fusion (b), the X₁ chromosome, which is derived from the ancestral X chromosome (c) and the X₂ chromosome, which is derived from the ancestral chromosome 9 (d). Scale bar, 5 μm

Fig. 2

The CENP-A antibody only localizes to one region on the Japan Sea neo-Y chromosome. Panels (a) and (c) show two independent metaphase spreads from Japan Sea embryos stained with the CENPA antibody (green) as well as the X and Y chromosome BAC 101E08 (purple) and the chromosome 9 BAC 35K20 (purple). Panels (b) and (d) are higher magnifications of the boxed regions in panels (a) and (c), highlighting the neo-Y chromosome with the two regions of BAC staining (purple arrowheads) flanking two distinct CENP-A puncta (white arrowhead). The CENP-A staining is located on the chromosome 9-derived part of the neo-Y chromosome. Panel (e) is a higher magnification of the boxed region in (a), highlighting the ancestral X₁ chromosome; 101E08 hybridizes to the middle of the long arm of X₁ (purple arrowhead), and there is one region of two CENP-A puncta (white arrowhead). Panel (f) is a higher magnification of the boxed region in (c), highlighting the unfused chromosome 9 (X₂); 35K20 hybridizes to the end of the long arm of X₂ (purple arrowhead), and there is one region of two CENP-A puncta (white arrowhead). Scale bar, 5 μm

Fig. 3

GacCEN staining is weak and variable on the ancestral Pacific Ocean male Y chromosome centromere. FISH with the GacGEN probe (green) and Y chromosome BAC 91G03 (purple) on four different metaphase spreads from the same Pacific Ocean male. Two metaphase spreads show GacCEN staining on the Y chromosome (a-d) while two metaphase spreads lack GacCEN staining on the Y chromosome (e-h). Panels (b, d) show higher magnification of the boxed regions in (a, c) highlighting Y chromosomes with weak GacCEN staining (green arrowhead), and panels (f, h) show higher magnification of the of the boxed regions in (e, g) highlighting Y chromosomes with no GacCEN staining. Scale bar, 5 μm

Fig. 4

GacCEN hybridizes to the centromere of chromosome 9 on the Japan Sea neo-Y chromosome. FISH with the GacCEN probe (green), Y chromosome BAC probe 188J19 (purple), and a chromosome 9 BAC probe 44L12 (purple) on a metaphase spread from a Japan Sea male is shown in panel (a). Panel (b) is a magnification of the neo-Y chromosome from the boxed region in panel (a). The GacGEN probe only localizes to the region of the ancestral chromosome 9 centromere on the neo-Y. Scale bar, 5 μm

Fig. 5

Mapping the regions flanking the ancestral Y centromere on the neo-Y fusion. FISH with the X and Y BACs 180J08 (green) and 171H24 (purple) on Japan Sea male interphase nuclei (a) and metaphase spreads (b). Panels (c) and (d) show higher magnifications of the boxed regions in panel (b). Both probes are present on the neo-Y (c) and the ancestral X₁ (d), although the probes appear to be closer together on the neo-Y (c) as compared to the ancestral X₁ (d). Scale bar, 5 μm

Fig. 6

GacCEN and centromere flanking regions on the Pacific Ocean X and Y, and Japan Sea neo-Y chromosomes. FISH with the X and Y BACs 180J08 and 171H24 (purple) and GacCEN (green) on a metaphase spread from a Pacific Ocean male is shown in panel (a) and a metaphase spread from a Japan Sea male in (d). Panels (b) and (c) are magnifications of the boxed regions in panel (a) showing two distinct regions of staining (purple arrowheads) flanking weak GacCEN staining (green arrowhead) on the unfused Y (b), and two distinct regions of staining (purple arrowheads) flanking strong GacCEN staining (green arrowhead) on the X (c). Panel (e) is a magnification of the boxed regions in panel (d) showing the two centromere flanking probes (purple arrowheads) are very close together on the neo-Y with no GacCEN staining in between. The strong GacCEN staining (green arrowhead) is from the chromosome 9 centromere on the neo-Y. Scale bar, 5 μm