Presentation1_Transcription and chromatin regulation by TAF4b during cellular quiescence of developing prospermatogonia.zip

Prospermatogonia (ProSpg) link the embryonic development of male primordial germ cells to the healthy establishment of postnatal spermatogonia and spermatogonial stem cells. While these spermatogenic precursor cells undergo the characteristic transitions of cycling and quiescence, the transcriptional events underlying these developmental hallmarks remain unknown. Here, we investigated the expression and function of TBP-associated factor 4b (Taf4b) in the timely development of quiescent mouse ProSpg using an integration of gene expression profiling and chromatin mapping. We find that Taf4b mRNA expression is elevated during the transition of mitotic-to-quiescent ProSpg and Taf4b-deficient ProSpg are delayed in their entry into quiescence. Gene ontology, protein network analysis, and chromatin mapping demonstrate that TAF4b is a direct and indirect regulator of chromatin and cell cycle-related gene expression programs during ProSpg quiescence. Further validation of these cell cycle mRNA changes due to the loss of TAF4b was accomplished via immunostaining for proliferating cell nuclear antigen (PCNA). Together, these data indicate that TAF4b is a key transcriptional regulator of the chromatin and quiescent state of the developing mammalian spermatogenic precursor lineage.</p

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

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

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

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