Dynamics of mono, di and tri-methylated histone H3 lysine 4 during male meiotic prophase I. Nuclei were co-stained for H3.1/H3.2. Progressing stages

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2 Dynamics of mono, di and tri-methylated histone H3 lysine 4 during male meiotic prophase I. Nuclei were co-stained for H3.1/H3.2. Progressing stages of spermatogenesis are shown from left to right. Arrows/dotted white lines indicate position and form of XY chromatin. Signals obtained for H3K4me1,2,3 in pachytene/diplotene spermatocytes and round spermatids were overall low when compared to spermatogonia, Sertoli cells and elongating spermatids (data not shown). a, Localisation of mono-methylated histone H3 lysine 4 (H3K4me1). The overall faint signal for H3K4me1 throughout the primary spermatocyte was lost during H3.1/H3.2 removal. During diplotene H3K4me1 accumulated in XY chromatin, eventually resulting in its enrichment in the condensed XY chromatin. In round spermatids this mark was faintly present throughout the chromatin. b, Localisation of di-methylated histone H3 lysine 4 (H3K4me2). As for H3K4me1, levels of this mark dropped during chromatin remodeling and eventually were lost from XY chromatin. XY levels were equal to autosomal levels in condensed meiotic chromosomes. In round spermatids the mark was present in the sex chromatin in a punctuated form. c, Localisation of tri-methylated histone H3 lysine 4 (H3K4me3). Prior to H3.1/H3.2 loss this mark was faintly present in sex chromatin. The mark disappeared completely from the sex body after H3.1/H3.2 removal. During the meiotic divisions a faint signal was detected on the sex chromosomes when compared to the signal autosomes showed. Histone H3K4me3 was present in the sex chromatin of round spermatids in levels equal to autosomal euchromatin.

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4 Dynamics of mono, di and tri-methylated histone H3 lysine 9 during male prophase I. Nuclei were co-stained for H3.1/H3.2. Progressing stages of spermatogenesis are shown from left to right. Arrows/dotted white lines indicate position and form of XY chromatin. a, Localisation of mono-methylated histone H3 lysine 9 (H3K9me1). XY levels of this mark varied from absent to low prior to H3.1/H3.2 removal. During H3.1/H3.2 loss (stages I-V), an increase of H3K9me1 was observed in the sex body, which persisted to early diplotene. However, in late diplotene (X-XI) this mark had disappeared again (its loss is likely a consequence of conversion to the di and triform, which are abundantly present at that stage). Condensed chromosomes exhibited faint staining. During haplophase overall nuclear staining for H3K9me1 was low. b, Localisation of di-methylated histone H3 lysine 9 (H3K9me2). Prior to H3.1/H3.2 removal enrichment of this mark was observed in XY chromatin. A sharp decrease of H3K9me2 was observed during and after removal of H3.1/H3.2. Signals gradually reappeared during pachytene stages VI-VIII. A further increase of levels was observed throughout diplotene. The condensed sex chromosomes were enriched for H3K9me2 and also in round spermatids this modification was prominent in the sex chromatin. c, Localisation of tri-methylated histone H3 lysine 9 (H3K9me3). Alike H3K9me2, this mark was enriched in XY chromatin prior to H3.1/H3.2 loss. Except for some faint signals observed in the heterochromatin of the X chromosome, H3K9me3 was lost from the sex body concomitant with H3.1/H3.2 loss. A gradual return of this mark in XY chromatin was observed during stages VI-XI. Condensed sex chromosomes exhibited high levels of H3K9me3 and also in the sex chromatin of round spermatids this mark was abundant.

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6 Dynamics of mono, di and tri-methylated histone H3 lysine 27 during male prophase I. Nuclei were co-stained for H3.1/H3.2. Progressing stages of spermatogenesis are shown from left to right. Arrows/dotted white lines indicate position and form of XY chromatin. a, Localisation of mono-methylated histone H3 lysine 27 (H3K27me1). High levels of H3K27me1 in the XY body were observed prior to H3.1/H3.2 loss. After H3.1/H3.2 loss a faint residual H3K27me1 staining was detected. This might be due to cross reactivity of this antibody with H3K9me1 and H4K20me1 (personal communication T. Jenuwein). Both of these marks were abundantly present in the XY body of pachytene spermatocytes but not when chromatin was condensed, when H3K27me1 was not detected also. In round spermatids H3K27me1 was again present in sex chromatin, though at a lower level than autosomal chromatin. b, Localisation of di-methylated histone H3 lysine 27 (H3K27me2). H3K27me2 was present in comparable levels in the XY chromosomes and autosomes in early pachytene spermatocytes. Together with H3.1/H3.2 this mark completely disappeared from XY chromatin, its absence from sex chromatin was sustained to round spermatids. c, Localisation of tri-methylated histone H3 lysine 27 (H3K27me3). Abundance of this modification was lower than H3K27me2 throughout the spermatocyte chromatin. Loss of H3.1/H3.2 resulted in the loss of this mark from the XY chromatin altogether. No return of this modification was observed into round spermatids.

7 Dynamics of mono and di-methylated histone H4 lysine 20 during male prophase I. Nuclei were co-stained for H3.1/H3.2. Progressing stages of spermatogenesis are shown from left to right. Arrows/dotted white lines indicate position and form of XY chromatin. a, Localisation of mono-methylated histone H4 lysine 20 (H4K20me1). Initial levels of H4K20me1 in the XY body were reduced when compared to autosomal levels. A rapid increase was observed at the onset of chromatin remodeling. However, a decline of signal was observed when the axial elements of the XYchromosomes started to bulge (diplotene, stages X-XI). Since conversion of H4K20me1 to H4K20me2 is limited and to H4K20me3 not observed at all, its loss

8 suggests the existence of an H4K20me1-demethylase. A reappearance of this mark was observed in the sex chromatin of round spermatids, where it was restricted to euchromatin. Together with our previous observation that H4K20me1 accumulates in sperm chromatin after gamete fusion, when a surge of histone deposition takes place 9, this suggests a function in nucleosome assembly. b, Localisation of di-methylated histone H4 lysine 20 (H4K20me2). Signals obtained for H4K20me2 were low in spermatocytes. An overall staining throughout the nucleus prior to H3.1/H3.2 loss was observed. Loss of signal from the XY chromosomes was detected during H3.1/H3.2 removal. In condensed chromosomes this mark became visible again in XY chromatin. However, in round spermatids (sex) chromatin did not exhibit staining.