Reduced MSY2 expression and diplotene arrest in <i>Taf4b</i>-deficient oocytes.

<p>(A) PND0 wild-type and <i>Taf4b</i>-deficient ovary tissue sections were stained with primary antibodies against MSY2 and germ cell marker TRA98, which were then quantified for the number of MSY2+/TRA98+ double-positive cells (B). As MSY2 indicates diplotene arrest, the data suggest a defect in diplotene arrival in <i>Taf4b</i>-deficient oocytes.</p

Reduced MLH1 meiotic recombination foci on pachytene chromosomes in <i>Taf4b</i>-deficient oocytes.

<p>(A) Oocyte meiotic chromosome spreads were prepared from E18.5 wild-type and <i>Taf4b</i>-deficient ovaries, and stained with primary antibodies against SYCP3 and MLH1. While wild-type oocytes have one or two intensely-stained MLH1 foci per homologous pair (white solid arrows), few of these foci are evident in <i>Taf4b</i>-deficient oocytes. Instead, the majority of MLH1 foci visible in <i>Taf4b</i>-deficient oocytes are comparatively faint (white dashed arrows) (B) Quantification of average MLH1 foci per oocyte. <i>Taf4b</i>-deficient oocytes possess significantly fewer total MLH1 foci than wild-type oocytes. *: n = 3 animals with 25 or more pachytene oocytes each, one-tailed t-test, p<0.0001</p

<i>Taf4b</i>-deficient oocytes experience defective meiotic progression and chromosome asynapsis.

<p>Pachytene spreads were prepared using the drying-down technique (50) on cell suspensions from E16.5 wild-type (A-D; I-L) and <i>Taf4b</i>-deficient (E-H; M-P) ovaries. Slides were stained for SYCP1 and SYCP3 (A-H), or SYCP3 and CENP-A (I-P). White arrowheads in (H) indicate regions of asynapsis in which SYCP3 is localized but SYCP1 is not; while white arrowheads in (P) indicate regions of asynapsis, many of which can be visualized by non-overlapping CENP-A centromeric foci. Slides were also stained for γH2AX and SYCP3 (Q, R) to visualize double-strand breaks. A high incidence of asynapsis (S), as scored by >20 CENP-A foci, as well as defects in meiotic progression (T), as scored by SYCP3 configuration (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006128#pgen.1006128.s001" target="_blank">S1 Fig</a>), were apparent in <i>Taf4b</i>-deficient oocytes. *: n = 4 animals each, one-tailed t-test, p<0.05</p

Model of TAF4b promoting a critical meiosis and oogenesis gene regulatory network: Data from chromatin immunoprecipitation experiments demonstrates that TAF4b occupies the proximal promoters of <i>Dazl</i>, <i>Stra8</i>, <i>Figlα</i>, and <i>Nobox</i>, ultimately resulting in their expression.

<p>Proper expression of these essential regulators facilitates expression of downstream meiosis and oogenesis genes, finally leading to the development of a healthy primordial follicle pool. Red arrows represent direct transcriptional regulation, gray arrows represent post-transcriptional regulation, and dashed lines indicate putative mechanisms.</p

Human <i>TAF4B</i> expression is highly correlated with the expression of meiotic regulators.

<p>(A) Ingenuity Pathway Analysis was performed on an existing data set profiling gene expression in human fetal ovary to determine coordinate regulation of genes with human <i>TAF4B</i>. The top twenty most-significant enrichments from this analysis revealed many functions related to meiotic regulation including <i>SYCP3</i>, <i>STAG3</i>, <i>YBX2</i>, and <i>DAZL</i>. (B) Pearson correlations were calculated for fertility genes of interest and R² values displayed.</p

TAF4b targets the promoters of critical meiosis and oogenesis regulators.

<p>(A) Wild-type E18.5 ovarian chromatin pulled down by antibodies against TAF4b or IgG and then qPCR-amplified using primers against the proximal promoters of <i>Stra8</i>, <i>Dazl</i>, <i>Figlα</i>, <i>Nobox</i> and a non-genic region upstream of <i>Nobox</i>. qPCR demonstrates enriched recovery of these proximal promoters with TAF4b-precipitated chromatin relative to IgG. (B) The <i>Nobox</i> proximal promoter, in particular, demonstrates enrichment for TAF4b, in contrast to a non-genic region upstream of the promoter. For all analyses, data from each primer set were normalized to the E18.5 mouse ovary genomic DNA input levels and represented as a percentage of that DNA input. Each qPCR reaction was performed in triplicate and averaged. Error bars indicate the normalized standard deviation resulting from experimental triplicate qPCR reactions.</p