Differential Expression and Sex Chromosome Association of CHD3/4 and CHD5 during Spermatogenesis

<div><p>ATP-dependent nucleosome remodelers of the CHD family play important roles in chromatin regulation during development and differentiation. The ubiquitously expressed CHD3 and CHD4 proteins are essential for stem cell function and serve to orchestrate gene expression in different developmental settings. By contrast, the closely related CHD5 is predominantly expressed in neural tissue and its role is believed to be restricted to neural differentiation. Indeed, loss of CHD5 contributes to neuroblastoma. In this study, we first demonstrate that CHD5 is a nucleosome-stimulated ATPase. We then compare CHD3/4 and CHD5 expression in mouse brain and show that CHD5 expression is restricted to a subset of cortical and hippocampal neurons whereas CHD3/4 expression is more widespread. We also uncover high levels of CHD5 expression in testis. CHD5 is transiently expressed in differentiating germ cells. Expression is first detected in nuclei of post-meiotic round spermatids, reaches a maximum in stage VIII spermatids and then falls to undetectable levels in stage IX spermatids. Surprisingly, CHD3/4 and CHD5 show complementary expression patterns during spermatogenesis with CHD3/4 levels progressively decreasing as CHD5 expression increases. In spermatocytes, CHD3/4 localizes to the pseudoautosomal region, the X centromeric region and then spreads into the XY body chromatin. In postmeiotic cells, CHD5 colocalises with macroH2A1.2 in association with centromeres and part of the Y chromosome. The subnuclear localisations of CHD4 and CHD5 suggest specific roles in regulation of sex chromosome chromatin and pericentromeric chromatin structure prior to the histone-protamine switch.</p></div

CHD5 is enriched in pericentromeric chromatin of round spermatids.

<p>(A, B) Spread nuclei of spermatids immunostained for CHD5 and counterstained with DAPI. An example of a round spermatid nucleus that is positive for CHD5 expression and displays the high intensity focus in the chromocenter is shown in A and such an example is also indicated with a white arrow in B, the yellow arrow indicates a spermatid nucleus that expresses CHD5, but does not have the extra CHD5 focus. The green arrow indicates a CHD5-negative round spermatid nucleus. (C) Spread nuclei of spermatids immunostained for CHD5 and H3K9me2, and counterstained with DAPI. The nuclear area encompassing the chromatin of the sex chromosome is indicated with a dashed line H3K9me2 marks the chromocenter and the sex chromosome in the left (early) round spermatid nucleus, and mainly the sex chromosome in the right (late) round spermatid nucleus. CHD5 level is very low in the left nucleus, but clearly enriched in the chromocenter and the focus (indicated with a white arrow) adjacent to the chromocenter and Y chromosome chromatin in the right nucleus.</p

macroH2A colocalizes with CHD3/4 in spermatocytes and with CHD5 in spermatids.

<p>(A,B) Spread wild type (A) and <i>Spo11</i> null (<i>Spo11^YF/YF</i>) (B) spermatocyte nuclei stained for MacroH2A.1 and CHD3/4. Colocalisation on the PAR and the X centromere is observed in pachytene, but not in diplotene nuclei of wild type spermatocytes. In the absence of functional SPO11, some zygotene-like nuclei show enrichment of both CHD4 and macroH2A.1 in the pseudo XY body. (C) Spread spermatid nucleus stained for CHD5, and MacroH2A, colocalisation of the CHD5/MacroH2A focus in the chromocenter is clear in the merge with DAPI (right image). (D) Spread spermatid nuclei stained with macroH2A (green) and CREST (red, left panel) or with CHD5 and CREST (right panel). The enlargements in the middle of each panel clearly show that the macroH2A.1/CHD5 spot in the chromocenter does not colocalise with CREST marking of the centromeres. (E) Spread spermatid nuclei costained for CHD5 and RNA polymerase II. Both high and low levels of RNA polymerase II can be observed in nuclei that express CHD5.</p

Expression of CHD3/4 and CHD5 is reciprocally regulated during spermatogenesis.

<p>(A) Western blot analysis CHD3/4, CHD5 and lamin expression in lysates from 3 week-old and from adult mice. Extracts from HEK cells expressing recombinant CHD5-Flag and extracts from murine brain were used as positive controls for CHD5 expression. Antibodies used for Western blot are indicated. (B) CHD5 protein is maximally expressed in round spermatids at Stages VII–VIII of the spermatognenic cycle. Upper images show results of the immunostaining and the bottom images show the PAS staining pattern of a section directly adjacent to the section shown above it. Left panel: low magnification (scale bars indicate 0.5 mm) of a mouse testis containing seminiferous tubules in different spermatogenic stages. Middle and right: higher magnification of the areas (scale bars indicate 0.05 mm) marked in the left panel, roman numerals indicate the stages of the spermatogenic cycle. (C) CHD5 is expressed in postmeiotic cells. Immunofluorescence analysis was performed using CHD5 antibody and visualized with confocal microscopy. Panel 1: Nuclei stained with DAPI. Panel 2: CHD5 staining, the outline of the tubule as seen in panel 1 is indicated with white lines. Panel 3: phosphorylated H2AX (γH2AX) staining. Spermatocytes in late meiotic prophase (pachytene and diplotene), show intense staining in a subregion of the nucleus (XY body) (arrow). Earlier spermatocytes (at the more basal part of the tubule) show nucleus wide γH2AX staining (arrowhead). Panel 4: overlay of the DAPI, CHD5 and γH2AX channels. (D) CHD5 and CHD3/4 expression at different stages of spermatogenesis. Panel 1: CHD5 is expressed in post-meiotic cells (100× magnification). Panel 2: CHD3/4 is expressed in the area where spermatogonia, Sertoli cells and spermatocytes are present (100× magnification). Panel 3: CHD5 immunostaining (250× magnification). Panel 4: CHD3/4 immunostaining (250x). Formalin-fixed paraffin-embedded tissue sections were immunostained with the antibodies indicated.</p

Localization patterns of CHD3/4 in spread nuclei of spermatocytes.

<p>Spread wild type mouse spermatocyte nuclei immunostained for CHD3/4 (green), SYCP3 (red), and counterstained with DAPI (blue). In wild type spermatocytes, CHD3/4 is most enriched on some ends of axial elements in zygotene (white arrows), then mainly on the pseudoautosomal region (PAR, P) in early (e) pachytene (see also enlargements at the bottom). Subsequently, all XY chromatin is somewhat enriched in mid (m) pachytene, followed by more specific restriction to the par and the X centromere in late (l) pachytene (enlargement on the bottom of the middle panel). In early and late diplotene, CHD4 enrichment is observed on centromeric heterochromatin, and more evenly distributed on the XY body (enlargement on the bottom of the right panel). XY bodies are indicated with dashed rectangles in the images. In the enlargements, X and Y indicate the axes corresponding to the X and Y chromosome, respectively.</p

CHD3/4 localisation in spermatocytes is independent of SPO11.

<p>(A) Spread wild type (left) and <i>Spo11</i> null (<i>Spo11^YF/YF</i>, right) spermatocytes stained for CHD3/4 (green), SYCP3 (red), and γH2AX (blue), the single CHD3/4 staining is shown in white to the right of each merge. In wild type nuclei, CHD3/4 does not colocalise with γH2AX in leptotene and zygotene. Specific foci accumulate on some chromosome ends (asterisks indicate examples), in particular on the pseudoautosomal region (PAR) of both X and Y, as can be observed in the late zygotene nucleus (white arrows). The inserted image shows part of the same nucleus with the X and Y chromosomal axes in white, for easy recognition. In pachytene, γH2AX and CHD3/4 colocalize on the XY body whereby CHD4 is particularly enriched on the PAR (white arrow) and the X centromere (asterisk). In <i>Spo11</i> null spermatocytes enrichment of CHD3/4 is observed on some chromosome ends in early zygotene (asterisks indicate examples), and in the pseudo XY body (white arrow) of later zygotene-like spermatocytes. (B) Spread <i>Spo11</i> null zygotene spermatocyte stained for SYCP3 and CHD3/4, and hybridized with a BAC probe recognizing the PAR. In the merge, an enlargement of the region encompassing the X and Y is shown (scale bar indicates 5 µm). In this nucleus CHD3/4 stain the PAR of the X and Y, but also mark the ends of 4 additional chromosome ends, that are also recognized, to a lesser extent, by the PAR probe. It is not clear what causes this crossreaction (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098203#s4" target="_blank">Materials and Methods</a>).</p

CHD5 protein is expressed in brain and testis.

<p>(A) CHD5 protein is expressed in mouse brain and testis. Western blot was performed with CHD5 antibody on lysates from different mouse tissues. Extracts from HEK cells transiently transfected with CHD5-Flag served as a positive control. Lamin B was used as a loading control. (B) Two different CHD5 isoforms are expressed in mouse brain and testis. Panel 1: schematic representation of the location of the PCR primers used to detect CHD5 cDNA of isoforms 1 and 2. Panel 2: schematic representation of part of the CHD5 gene structure showing the two different putative splicing variants. Exons 23, 24 and 25 are indicated. Horizontal lines are introns. Diagonal lines indicate splicing. Panel 3: RT-PCR detecting expression of different CHD5 isoforms in mouse testis and brain. H₂O: negative control. Asterisk: primer dimers.</p

CHD5 is a nucleosome stimulated ATPase.

<p>(A) Schematic drawing of CHD3, CHD4 and CHD5 proteins showing conserved domains and the CHD5 specific sequence. (B) Table showing sequence identity between different domains of CHD3, CHD4 and CHD5. (C) Expression of recombinant CHD5. Recombinant CHD5-Flag was purified from extracts of baculovirus-infected Sf9 cells. Extracts were incubated with anti-Flag affinity resin, washed and eluted with an excess of Flag peptide. Purified protein was seperated by SDS-PAGE followed by Western blotting with CHD5 antibody (lane 1) or silver staining (lane 2). Asterisk indicates degradation products with intact anti-CHD5 epitope. (D) CHD5 is a nucleosome-stimulated ATPase. Purified recombinant CHD5-Flag was subjected to ATPase assay as described in Materials and Methods. Different amounts of recombinant human CHD5-Flag, naked DNA or <i>in vitro</i> assembled nucleosomes were used as indicated at the bottom of the panel. Reaction products were separated by thin layer chromatography and the amount of released ³²P_i was quantitated. The percentage of ATP hydrolysis was calculated for each reaction. The percentage of ATP hydrolysis of control reactions (without enzyme) was set to one. Error bars depict the standard deviation of 3 independent experiments.</p