Localisation of the SMC loading complex Nipbl/Mau2 during mammalian meiotic prophase I

Abstract

Evidence from lower eukaryotes suggests that the chromosomal associations of all the structural maintenance of chromosome (SMC) complexes, cohesin, condensin and Smc5/6, are influenced by the Nipbl/Mau2 heterodimer. Whether this function is conserved in mammals is currently not known. During mammalian meiosis, very different localisation patterns have been reported for the SMC complexes, and the localisation of Nipbl/Mau2 has just recently started to be investigated. Here, we show that Nipbl/Mau2 binds on chromosomal axes from zygotene to mid-pachytene in germ cells of both sexes. In spermatocytes, Nipbl/Mau2 then relocalises to chromocenters, whereas in oocytes it remains bound to chromosomal axes throughout prophase to dictyate arrest. The localisation pattern of Nipbl/Mau2, together with those seen for cohesin, condensin and Smc5/6 subunits, is consistent with a role as a loading factor for cohesin and condensin I, but not for Smc5/6. We also demonstrate that Nipbl/Mau2 localises next to Rad51 and γH2AX foci. NIPBL gene deficiencies are associated with the Cornelia de Lange syndrome in humans, and we find that haploinsufficiency of the orthologous mouse gene results in an altered distribution of double-strand breaks marked by γH2AX during prophase I. However, this is insufficient to result in major meiotic malfunctions, and the chromosomal associations of the synaptonemal complex proteins and the three SMC complexes appear cytologically indistinguishable in wild-type and Nipbl (+/-) spermatocytes.

Fig. 1

Chromosomal localisation of Nipbl/Mau2 in prophase I spermatocytes. a Testicular nuclear spreads were stained with rabbit anti-Sycp3 (green), guinea pig anti-Nipbl (red) and human anti-CREST (white), and images were staged according to established Sycp3 and CREST-staining patterns during prophase I. Nipbl was found on chromosomal axes from leptotene until mid-pachytene, when it translocated to chromocentres. b Using identical imaging settings, Nipbl staining of individual nuclei were quantitated, subtracting the background intensity of a neighbouring empty area. Nipbl staining was three- to fourfold more intense during late pachytene/diplotene stages than at earlier stages. c Testicular nuclear spreads, stained with rabbit anti-Mau2 (green), guinea pig anti-Nipbl (red) and human anti-CREST (white)

Fig. 2

Chromosomal localisation of Nipbl/Mau2 in prophase I oocytes. Developing oocytes were isolated from female embryos at E16.5 to E19.5 and stained with rabbit anti-Sycp3 (green), guinea pig anti-Nipbl (red) and human anti-CREST (white). Images were staged through meiosis prophase I as in Fig. 1. Nipbl binds chromosomal axes throughout the meiotic prophase

Fig. 3

Mau2 and cohesin co-localise between zygotene and early pachytene in spermatocytes. Testicular nuclear spreads were stained with rabbit anti-Mau2 (red), guinea pig anti-SMC1β (green) and human anti-CREST (white). Images were staged through meiosis prophase I as in Fig. 1

Fig. 4

Reduced Nipbl spermatocyte expression and increased DNA damage sensitivity in Nipbl ^+/− MEFs. a Western blot showing reduced Nipbl expression in testis from Nipbl ^+/− animals using the same antibody as for immunofluorescence. b Wild-type and Nipbl ^+/− embryonic fibroblasts were exposed to the indicated radiation doses and allowed to form colonies. c Immunofluorescent staining of rabbit anti-Smc3 (green), guinea pig anti-Nipbl (red) and human anti-CREST (white) in prophase I spermatocytes from wild-type and Nipbl ^+/− mice. Top row: Nipbl was first detected in leptotene/zygotene stages in wild type or later in zygotene in Nipbl ^+/− spermatocytes. Second row: In late zygotene, Nipbl appeared to be lost from axial elements in Nipbl ^+/− spermatocytes. Middle row: Mid-pachytene spermatocytes displayed accumulation of Nipbl on chromocentres in both wild type and Nipbl ^+/−, and Smc3 binding to chromosomal axes appeared normal. Fourth row: In late pachytene/diplotene spermatocytes, Nipbl was fully localised to chromocentres. Bottom row: Diplotene spermatocytes displaying progressively weaker Smc3 staining and full labelling of chromocentres by Nipbl. Images were staged through meiosis prophase I as in Fig. 1

Fig. 5

Influence of the SC components Sycp1 and 3 on Nipbl/Mau2 localisation. Nuclear spreads of embryonic ovaries or testes stained with rabbit anti-Mau2 (red), guinea pig anti-Smc1β (green, left), guinea pig anti-Stag3 (green, right) and human anti-CREST (white) in wild-type and Sycp1 ^−/− or Sycp3 ^−/− mice as indicated. Germ cells from Sycp1 ^−/− and Sycp3 ^−/− are arrested in a pre-pachytene state. Wild-type pachytene oocytes (left) and zygotene spermatocytes (right) are shown for comparison

Fig. 6

Nipbl and condensin I co-localise between zygotene and mid-pachytene in spermatocytes, but condensin staining is not affected by Nipbl haploinsufficiency. Testicular nuclear spreads of wild-type and Nipbl ^+/− spermatocytes were stained with rabbit anti-Cap-G (green), guinea pig anti-Nipbl (red) and human anti-CREST (white)

Fig. 7

No apparent co-localisation between Nipbl and Smc5/6 in spermatocytes. Testicular nuclear spreads were stained with rabbit anti-Smc6 (green), guinea pig anti-Nipbl (red) and human anti-ACA (white)

Fig. 8

Nipbl does not co-localise with markers for DNA damage and repair. Testicular nuclear spreads were stained with guinea pig anti-Nipbl (red), mouse anti-γH2AX (green in first three columns) and mouse anti-RAD51 (green in fourth column)

Fig. 9

Pachytene Nipbl ^+/− spermatocytes display diffuse and disorganised staining of γH2AX. Immunofluorescent staining of Sycp3 (green), Nipbl (red) and γH2AX (white). Images were staged according to the distribution of Sycp3 and Nipbl along chromosomal axes. In late zygotene and early pachytene (top row), most of the ubiquitous γH2AX of early prophase signals disappear from the nucleus, except at the sex body, which appears in as an irregular elongated shape. In wild type, one can observe residual γH2AX signals organised at chromosome axes, while the γH2AX outside of the sex body in Nipbl ^+/− are more prominent and distributed all over the nucleus. In early and mid-pachytene, when Nipbl starts to re-localise to chromocentres (middle and bottom rows), residual γH2AX staining is still stronger in Nipbl ^+/− than in wild type

Fig. 10

Spatial and temporal connection between Nipbl and H3K9me3 re-localisation to heterochromatin. Testicular nuclear spreads were stained with rabbit anti-H3K9me3 (green), guinea pig anti-Nipbl (red) and human anti-CREST (white). In early pachytene cells (upper row), H3K9me3 is seen on chromocentres and as a diffuse nuclear staining. During mid-pachytene (middle rows), H3K9me3 persists on chromocentres, while the diffuse nuclear staining fades concomitantly as Nipbl relocates to heterochromatin. In diplotene, both Nipbl and H3K9me3 can only be detected on chromocentres (bottom row)