RNA polymerase II pausing is essential during spermatogenesis for appropriate gene expression and completion of meiosis.

Male germ cell development requires precise regulation of gene activity in a cell-type and stage-specific manner, with perturbations in gene expression during spermatogenesis associated with infertility. Here, we use steady-state, nascent and single-cell RNA sequencing strategies to comprehensively characterize gene expression across male germ cell populations, to dissect the mechanisms of gene control and provide new insights towards therapy. We discover a requirement for pausing of RNA Polymerase II (Pol II) at the earliest stages of sperm differentiation to establish the landscape of gene activity across development.

RNA polymerase II pausing is essential during spermatogenesis for appropriate gene expression and completion of meiosis.

Male germ cell development requires precise regulation of gene activity in a cell-type and stage-specific manner, with perturbations in gene expression during spermatogenesis associated with infertility. Here, we use steady-state, nascent and single-cell RNA sequencing strategies to comprehensively characterize gene expression across male germ cell populations, to dissect the mechanisms of gene control and provide new insights towards therapy. We discover a requirement for pausing of RNA Polymerase II (Pol II) at the earliest stages of sperm differentiation to establish the landscape of gene activity across development.

Dynamic control of chromatin-associated mA methylation regulates nascent RNA synthesis.

N-methyladenosine (mA) methylation is co-transcriptionally deposited on mRNA, but a possible role of mA on transcription remains poorly understood. Here, we demonstrate that the METTL3/METTL14/WTAP mA methyltransferase complex (MTC) is localized to many promoters and enhancers and deposits the mA modification on nascent transcripts, including pre-mRNAs, promoter upstream transcripts (PROMPTs), and enhancer RNAs. PRO-seq analyses demonstrate that nascent RNAs originating from both promoters and enhancers are significantly decreased in the METTL3-depleted cells.

Differential Occupancy of Two GA-Binding Proteins Promotes Targeting of the Drosophila Dosage Compensation Complex to the Male X Chromosome.

Little is known about how variation in sequence composition alters transcription factor occupancy to precisely recruit large transcription complexes. A key model for understanding how transcription complexes are targeted is the Drosophila dosage compensation system in which the male-specific lethal (MSL) transcription complex specifically identifies and regulates the male X chromosome. The chromatin-linked adaptor for MSL proteins (CLAMP) zinc-finger protein targets MSL to the X chromosome but also binds to GA-rich sequence elements throughout the genome.

Drosophila Dosage Compensation Loci Associate with a Boundary-Forming Insulator Complex.

Chromatin entry sites (CES) are 100- to 1,500-bp elements that recruit male-specific lethal (MSL) complexes to the X chromosome to upregulate expression of X-linked genes in male flies. CES contain one or more ∼20-bp GA-rich sequences called MSL recognition elements (MREs) that are critical for dosage compensation. Recent studies indicate that CES also correspond to boundaries of X-chromosomal topologically associated domains (TADs).

Expansion of GA Dinucleotide Repeats Increases the Density of CLAMP Binding Sites on the X-Chromosome to Promote Drosophila Dosage Compensation.

Dosage compensation is an essential process that equalizes transcript levels of X-linked genes between sexes by forming a domain of coordinated gene expression. Throughout the evolution of Diptera, many different X-chromosomes acquired the ability to be dosage compensated. Once each newly evolved X-chromosome is targeted for dosage compensation in XY males, its active genes are upregulated two-fold to equalize gene expression with XX females.