Human sperm displays rapid responses to diet.

The global rise in obesity and steady decline in sperm quality are two alarming trends that have emerged during recent decades. In parallel, evidence from model organisms shows that paternal diet can affect offspring metabolic health in a process involving sperm tRNA-derived small RNA (tsRNA). Here, we report that human sperm are acutely sensitive to nutrient flux, both in terms of sperm motility and changes in sperm tsRNA. Over the course of a 2-week diet intervention, in which we first introduced a healthy diet followed by a diet rich in sugar, sperm motility increased and stabilized at high levels. Small RNA-seq on repeatedly sampled sperm from the same individuals revealed that tsRNAs were up-regulated by eating a high-sugar diet for just 1 week. Unsupervised clustering identified two independent pathways for the biogenesis of these tsRNAs: one involving a novel class of fragments with specific cleavage in the T-loop of mature nuclear tRNAs and the other exclusively involving mitochondrial tsRNAs. Mitochondrial involvement was further supported by a similar up-regulation of mitochondrial rRNA-derived small RNA (rsRNA). Notably, the changes in sugar-sensitive tsRNA were positively associated with simultaneous changes in sperm motility and negatively associated with obesity in an independent clinical cohort. This rapid response to a dietary intervention on tsRNA in human sperm is attuned with the paternal intergenerational metabolic responses found in model organisms. More importantly, our findings suggest shared diet-sensitive mechanisms between sperm motility and the biogenesis of tsRNA, which provide novel insights about the interplay between nutrition and male reproductive health.

High-Affinity Sites Form an Interaction Network to Facilitate Spreading of the MSL Complex across the X Chromosome in Drosophila.

Dosage compensation mechanisms provide a paradigm to study the contribution of chromosomal conformation toward targeting and spreading of epigenetic regulators over a specific chromosome. By using Hi-C and 4C analyses, we show that high-affinity sites (HAS), landing platforms of the male-specific lethal (MSL) complex, are enriched around topologically associating domain (TAD) boundaries on the X chromosome and harbor more long-range contacts in a sex-independent manner. Ectopically expressed roX1 and roX2 RNAs target HAS on the X chromosome in trans and, via spatial proximity, induce spreading of the MSL complex in cis, leading to increased expression of neighboring autosomal genes. We show that the MSL complex regulates nucleosome positioning at HAS, therefore acting locally rather than influencing the overall chromosomal architecture. We propose that the sex-independent, three-dimensional conformation of the X chromosome poises it for exploitation by the MSL complex, thereby facilitating spreading in males.

Generation of reporter plasmids containing defined base modifications in the DNA strand of choice.

Physiological effects of DNA bases other than A, G, C, and T as well as ways of removal of such bases from genomes are studied intensely. Methods for targeted insertion of modified bases into DNA, therefore, are highly demanded in the fields of DNA repair and epigenetics. This article describes efficient procedures for incorporation of modified DNA bases into a plasmid-borne enhanced green fluorescent protein (EGFP) gene. The procedure exploits excision of a stretch of 18 nt from either the transcribed or nontranscribed DNA strand with the help of the sequence-specific nicking endonucleases Nb.Bpu10I and Nt.Bpu10I. The excised single-stranded oligonucleotide is then swapped for a synthetic DNA strand containing a desired base modification. Base modifications that form Watson-Crick-type base pairs were efficiently incorporated into plasmid DNA by a straightforward strand exchange, which was achieved by local melting in the presence of large excesses of the synthetic oligonucleotides and reannealing followed by ligation. Base modifications that cause significant distortions of the normal DNA structure, such as thymine glycol and uracil mispaired with guanine, failed to produce high yields of direct strand exchange but could still be incorporated very efficiently when the excised fragment was depleted in an intermediate step.

Mouse CSB protein is important for gene expression in the presence of a single-strand break in the non-transcribed DNA strand.

CSB protein is required for strand-specific repair of bulky DNA lesions in transcribed genes and mediates transcription recovery after exposure to DNA-damaging agents. We enzymatically generated DNA single-strand breaks (SSBs) with 3'-OH and 5'-phosphate termini in defined positions of a plasmid-borne gene and measured their effect on transcription in cell lines with different statuses of the Csb gene. A single SSB in the transcribed region of the gene caused significant decrease of gene expression. In all tested cell lines of mouse and human origin, a SSB in the transcribed DNA strand was less harmful for gene expression than a SSB situated in the opposing DNA strand. CSB deficiency exhibited no effect on the expression of the nicked DNA in human fibroblasts immortalised by SV40 large T-antigen but caused a very strong decrease of gene expression in spontaneously transformed mouse embryonic fibroblasts (MEFs). Compared to the corresponding CSB-proficient MEFs, the effect was on average 6.7-fold stronger for a defined SSB located in the non-transcribed DNA strand, but only 2.4-fold for a SSB in the transcribed strand and 1.7-fold for a SSB located in the non-genic region. At the same time, CSB deficiency did not compromise the overall efficiency of repair of SSBs generated by treatment of the cells with hydrogen peroxide. The gene expression data thus indicate that CSB prevents irreversible transcription failures at the sites of DNA damage, acting preferentially at SSBs located in the non-transcribed DNA strand of the transcribed genes. We further conclude that SSBs in the non-transcribed DNA strand are commonly more harmful for transcription than those situated in the transcribed strand.