Sequential changes at differentiation gene promoters as they become active in a stem cell lineage

Abstract

SUMMARYTranscriptional silencing of terminal differentiation genes by the Polycomb group (PcG) machinery is emerging as a key feature of precursor cells in stem cell lineages. How, then, is this epigenetic silencing reversed for proper cellular differentiation? Here, we investigate how the developmental program reverses local PcG action to allow expression of terminal differentiation genes in the Drosophila male germline stem cell (GSC) lineage. We find that the silenced state, set up in precursor cells, is relieved through developmentally regulated sequential events at promoters once cells commit to spermatocyte differentiation. The programmed events include global downregulation of Polycomb repressive complex 2 (PRC2) components, recruitment of hypophosphorylated RNA polymerase II (Pol II) to promoters, as well as the expression and action of testis-specific homologs of TATA-binding proteinassociated factors (tTAFs). In addition, action of the testis-specific meiotic arrest complex (tMAC), a tissue-specific version of the MIP/dREAM complex, is required both for recruitment of tTAFs to target differentiation genes and for proper cell type-specific localization of PRC1 components and tTAFs within the spermatocyte nucleolus. Together, the action of the tMAC and tTAF cell type-specific chromatin and transcription machinery leads to loss of Polycomb and release of stalled Pol II from the terminal differentiation gene promoters, allowing robust transcription.

Drosophila danyis essential for transcriptional control and nuclear architecture in spermatocytes

“…Indeed, some important chromatin regulators and nuclear envelope proteins have been shown to undergo drastic changes in expression levels during normal spermatogenesis. Su(Hw), a multi-zinc finger DNA-binding protein that can function as a transcriptional insulator and modulator of chromatin association with the nuclear lamina (Soshnev et al, 2013;van Bemmel et al, 2010), and the PRC2 complex components E(z) and Su(z) (Chen et al, 2011) are strongly downregulated in early spermatocytes, whereas Lamin C (LamC) expression is induced (Chen et al, 2013). However, in these particular cases, we did not detect an abnormal expression program in dany mutant testis by immunolabeling (data not shown).…”

“…S4A). tTAFs have been proposed to function in part by sequestering Polycomb (Pc), a core component of the PRC1 repressive chromatin complex (Steffen and Ringrose, 2014), in the nucleolus (Chen et al, 2005(Chen et al, , 2011White-Cooper and Caporilli, 2013), although this notion has been challenged recently (ElSharnouby et al, 2013). We were unable to detect abnormalities in the expression and localization of Pc-EGFP in dany mutants (Fig.…”

MicroRNAs downregulate Bag of marbles to ensure proper terminal differentiation in the Drosophila male germline

“…Testes cDNA preparation and RT-PCR were performed as described previously (Chen et al, 2011). Each PCR reaction was performed in duplicates and Ct numbers were averaged, at least three independent biological replicates were used.…”

Chromatin Immunoprecipitation (ChIP) using <em>Drosophila</em> tissue

“…To compare the histone modification level and gene expression, we used a python script which has been described in a previous publication 3 . Figure 1 4 . In bam testes, there is a failure in the transition from proliferative spermatogonia to differentiating spermatocytes 5,6 .…”

“…These genes are highly enriched with the repressive H3K27me3 histone modification 7 (Figure 1A), but are devoid of the active H3K4me3 histone modification 8 (Figure 1B), a chromatin signature we called 'monovalent' 7 . Enrichment of either H3K27me3 or H3K4me3 is determined by normalization to a constitutively expressed Cyclin A (CycA) gene 4 .…”