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Inactivation or non-reactivation: what accounts better for the silence of sex chromosomes during mammalian male meiosis?

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Abstract

During the first meiotic prophase in male mammals, sex chromosomes undergo a program of transcriptional silencing called meiotic sex chromosome inactivation (MSCI). MSCI is triggered by accumulation of proteins like BRCA1, ATR, and γH2AX on unsynapsed chromosomes, followed by local changes on the sex chromatin, including histone modifications, incorporation of specific histone variants, non-histone proteins, and RNAs. It is generally thought that MSCI represents the transition of unsynapsed chromatin from a transcriptionally active state to a repressed state. However, transcription is generally low in the whole nucleus during the early stages of the first meiotic prophase, when markers of MSCI first appear, and is then reactivated globally during pachytene. Thus, an alternative possibility is that MSCI represents the targeted maintenance and/or reinforcement of a prior repressed state, i.e., a failure to reactivate. Here, we present an analysis of the temporal and spatial appearance of transcriptional and MSCI markers, as well as chromatin modifications related to transcriptional regulation. We show that levels of RNA pol II and histone H3 acetylated at lysine 9 (H3K9ac) are low during leptotene, zygotene, and early pachytene, but increase strongly in mid-pachytene, indicating that reactivation occurs with some delay after synapsis. However, while transcription markers appear abundantly on the autosomes at mid-pachytene, they are not directed to the sex chromosomes. Interestingly, we found that chromatin modifications related to transcriptional silencing and/or MSCI, namely, histone H3 trimethylated at lysine 9 (H3K9me3), histone H3 monomethylated at lysine 4 (H3K4me1), γH2AX, SUMO1, and XMR, appear on the sex chromosomes before autosomes become reactivated. These results suggest that the onset of MSCI during late zygotene and early pachytene may prevent sex chromosome reactivation during mid-pachytene instead of promoting inactivation de novo. Additionally, we found temporal differences between the X and Y chromosomes in the recruitment of DNA repair and MSCI markers, indicating a differential regulation of these processes. We propose that many of the meiotic defects attributed to failure to silence sex chromosomes could be interpreted as a more general process of transcriptional misregulation that occurs under certain pathological circumstances in zygotene and early pachytene.

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Acknowledgements

We express our sincere thanks to Denise Escalier (Université Paris 5, Paris, France) for providing the XMR antibody and to Dr. Teresa L. Mastracci and Dr. Juan Luis Santos (Universidad Complutense de Madrid) for their critical review of the manuscript. This work was supported by grant BFU2009-10987 from Ministerio de Ciencia e Innovación, grants A/017762/08 and A/023249/09 from Agencia Española de Cooperación Internacional para el Desarrollo (Spain), and FONDECYT grant 1080090 (Chile).

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  1. Marcia Manterola

    Present address: Department of Genetics and Development, Columbia University Medical Center, New York, NY, USA

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  1. Unidad de Biología Celular, Departamento de Biología, Universidad Autónoma de Madrid, Madrid, Spain

    Jesús Page, Roberto de la Fuente, María Teresa Parra, Alberto Viera & Julio S. Rufas

  2. Programa de Genética Humana, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile

    Marcia Manterola, Soledad Berríos & Raúl Fernández-Donoso

  3. Laboratorio C-001, Departamento de Biología, Edificio de Ciencias Biológicas, Universidad Autónoma de Madrid, C/Darwin Nº2, Cantoblanco, 28049, Madrid, Spain

    Jesús Page

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Supplementary Figure 1

Location of SCP3 (green), centromeres (red) and γH2AX (blue) during late zygotene. (A). Most autosomes are still undergoing synapsis. γH2AX is present in both synapsed and unsynapsed autosomes. The centromeres are located in distal position in all autosomes. The X chromosome (enlarged in B) also presents a centromere located at one end of its AE (arrowhead). The opposite end has a thickening corresponding to the PAR (arrow). This chromosome is completely labeled with γH2AX, except in the pericentromeric region. The Y chromosome (enlarged in C) presents its centromere located in a sub-distal position (arrowhead), thus showing a visible short arm. This chromosome shows a thickening of the AE at the opposite end (arrow), corresponding with the PAR. γH2AX labeling is restricted to one chromosome end, presumably stemming from the transition between the thickened and the regular region of its AE. (JPEG 31 kb)

High resolution image (TIFF 2504 kb)

Supplementary Figure 2

DAPI staining of sex chromosomes in the same cells shown in Figure 1. A distinct conformation of the sex chromatin is not detected during zygotene (A–B) or early pachytene (C–D). A different conformation is only observed from mid pachytene onwards (E–L). rb indicates the round body, which is usually associated with the sex chromosomes from mid pachytene onwards. (JPEG 64 kb)

High resolution image (TIFF 2538 kb)

Supplementary Figure 3

Location of SCP3 (green) and γH2AX (red) during mid and late prophase-I. (A–C). Early-mid pachytene. All the autosomes are completely synapsed and sex chromosomes also show a great extension of synapsis. When exposure of the picture is adjusted to the intensity of the labeling on the sex chromosomes (A) γH2AX is not detectable in the chromatin of autosomes. However, if the picture is over exposed (B–C), an intense labeling emerges on the chromatin of all autosomal bivalents. This is mostly restricted to the chromatin around the LEs (arrowheads) and clearly does not involve the LEs (see enlarged detail in the upper insert). Additionally, some bivalents show great γH2AX signals that emanate from the LEs and seem to expand to greater chromatin loops (arrow). (D–F). Mid pachytene. γH2AX labeling on the sex chromosomes now depicts a more regular border of the sex body. Upon over-exposure of the pictures (E–F) the labeling on the autosomes is still visible in the chromatin close to the LEs, but γH2AX is now concentrated in some specific regions along bivalents, yielding a banded-like pattern (arrows). (G–I). Late pachytene. The sex body remains intensely labeled with γH2AX. The labeling on the autosomes in now restricted to a few spots (arrows) located over some but not all bivalents. (J–L). Diplotene. γH2AX labeling of the sex body is still intense. No γH2AX in detected on the autosomes at this stage. (JPEG 103 kb)

High resolution image (TIFF 6322 kb)

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Page, J., de la Fuente, R., Manterola, M. et al. Inactivation or non-reactivation: what accounts better for the silence of sex chromosomes during mammalian male meiosis?. Chromosoma 121, 307–326 (2012). https://doi.org/10.1007/s00412-012-0364-y

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