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. 2009 Aug;19(8):1338-49.
doi: 10.1101/gr.094953.109. Epub 2009 Jul 7.

Endonuclease-sensitive regions of human spermatozoal chromatin are highly enriched in promoter and CTCF binding sequences

Ali Arpanahi  1 , Martin Brinkworth, David Iles, Stephen A Krawetz, Agnieszka Paradowska, Adrian E Platts, Myriam Saida, Klaus Steger, Philip Tedder, David Miller
Affiliations

Endonuclease-sensitive regions of human spermatozoal chromatin are highly enriched in promoter and CTCF binding sequences

Ali Arpanahi et al. Genome Res. 2009 Aug.

Abstract

During the haploid phase of mammalian spermatogenesis, nucleosomal chromatin is ultimately repackaged by small, highly basic protamines to generate an extremely compact, toroidal chromatin architecture that is critical to normal spermatozoal function. In common with several species, however, the human spermatozoon retains a small proportion of its chromatin packaged in nucleosomes. As nucleosomal chromatin in spermatozoa is structurally more open than protamine-packaged chromatin, we considered it likely to be more accessible to exogenously applied endonucleases. Accordingly, we have used this premise to identify a population of endonuclease-sensitive DNA sequences in human and murine spermatozoa. Our results show unequivocally that, in contrast to the endonuclease-resistant sperm chromatin packaged by protamines, regions of increased endonuclease sensitivity are closely associated with gene regulatory regions, including many promoter sequences and sequences recognized by CCCTC-binding factor (CTCF). Similar differential packaging of promoters is observed in the spermatozoal chromatin of both mouse and man. These observations imply the existence of epigenetic marks that distinguish gene regulatory regions in male germ cells and prevent their repackaging by protamines during spermiogenesis. The ontology of genes under the control of endonuclease-sensitive regulatory regions implies a role for this phenomenon in subsequent embryonic development.

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Figures

Figure 1.
Figure 1.
Analysis of DNA fractions obtained by SRD and MND treatment of sperm nuclei. All DNA samples were resolved on 1.8% agarose gels. (Lane 1) A 0.4–10-kb DNA ladder. (Lane 2) Total (unfractionated) DNA (NF). (Lane 3) The soluble DNA released after extraction of sperm nuclei with 0.65 M NaCl followed by digestion with BamHI and EcoRI (SRDS). (Lane 5) The corresponding insoluble pellet (SRDI). (Lane 4) DNA released from sperm after MNase digestion (MNDS). (Lane 6) The corresponding insoluble pellet (MNDI).
Figure 2.
Figure 2.
Whole chromosome CGH plots (human). (A,C,E,G) The log2R moving average profiles obtained from the 44K CGH analysis of SRDS/SRDI fraction pairs of three individual men (44K1, 44K2, and 44K3) for chromosomes 11, 12, 16, and 19. The moving average profile from an additional fraction pair analyzed on the 244K CGH platform is also shown. (B,D,F,H) The 244K probe plots from SRDS/SRDI and MNDS/MNDI data as individual log2R values >0.5; (green) soluble (S); (red) ≤0.5 insoluble (I) fractions aligned alongside Ensembl gene density profiles and chromosome ideograms (http://www.ensembl.org). Moving average traces that are dynamically equivalent to those drawn in panels A, C, E, and G are drawn between the fraction pairs. (Arrowheads) Olfactory receptor gene clusters on chromosomes 11 and 12 and zinc finger clusters on chromosome 19. With the exception of these cluster types, the tendency for the SRDS/MNDS probes to correspond closely with gene density profiles is clearly evident.
Figure 3.
Figure 3.
Whole chromosome CGH scatterplots (mouse). The log2R moving averages from murine MNDS/MNDI data generated on the Agilent mouse 244K CGH platform are shown for chromosome 2, chromosome 5 (B), chromosome 7 (C), and chromosome 17 (D). The probe profiles from MNDS/MNDI fractions as individual log2R values >0.5; (green) soluble (S); (red) ≤0.5 insoluble (I) are aligned alongside Ensembl gene density profiles and chromosome ideograms. As with the human data, the tendency for the MNDS probes to correspond closely with gene density profiles is evident. (Arrowhead) A prominent olfactory receptor gene cluster on chromosome 2 where this trend (as in human sperm chromatin) is reversed.
Figure 4.
Figure 4.
Probe signal intensities from MND and SRD fractions. Boxplots showing the range for all probes in all fractions with at least a twofold change in probe signal intensity from the median value (black bars). The plots show clearly that the highest numbers of high signal intensities are in the soluble fractions.
Figure 5.
Figure 5.
Gene regulatory sequences in MND and SRD fractions. Boxplots showing the partitioning behavior of sequences detected by all probes located within promoters (P all) in the SRD (A) and MND (B) fractions compared with those within genic (G) and intergenic (IG) sequences. The partitioning behavior of sequences detected by probes within CTCF clusters in association with (C+P+) or without (C+P−) promoters is shown for SRD (C) and MND (D) fractions compared with the behavior of promoters alone (C−P+; equivalent to “P all” in A and B) or the absence of both promoters and CTCF clusters (C−P−; equivalent to G/IG in A and B). The center of the boxes indicates the median value with the first and third quartiles drawn on either side. Notches indicate significant differences in median values (Tukey's honest significance difference). In all plots, log2R values >0 indicate solubility and <0 indicate insolubility. Note that the greatest skew toward solubility is for probes detecting sequences located within promoters and CTCF clusters, followed by probes with CTCF clusters alone, and then within promoters alone.
Figure 6.
Figure 6.
Regions flanking sites of CTCF binding are also enriched in soluble chromatin. The plot shows that the strong preference for CTCF clusters to partition into soluble SRD and MND fractions is shared by sequences flanking CTCF binding sites and extends for several kilobases. Note that median log2R values only return toward the median for the entire data sets after 50 kb and that sequences detected by probes lying in regions more than 75 kb from any sites of CTCF binding are actually enriched in insoluble chromatin (Kruskal-Wallis; χ2 = 534.5, df = 9, P < 0.0001).
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