Paternal Poly (ADP-ribose) Metabolism Modulates Retention of Inheritable Sperm Histones and Early Embryonic Gene Expression

To achieve the extreme nuclear condensation necessary for sperm function, most histones are replaced with protamines during spermiogenesis in mammals. Mature sperm retain only a small fraction of nucleosomes, which are, in part, enriched on gene regulatory sequences, and recent findings suggest that these retained histones provide epigenetic information that regulates expression of a subset of genes involved in embryo development after fertilization. We addressed this tantalizing hypothesis by analyzing two mouse models exhibiting abnormal histone positioning in mature sperm due to impaired poly(ADP-ribose) (PAR) metabolism during spermiogenesis and identified altered sperm histone retention in specific gene loci genome-wide using MNase digestion-based enrichment of mononucleosomal DNA. We then set out to determine the extent to which expression of these genes was altered in embryos generated with these sperm. For control sperm, most genes showed some degree of histone association, unexpectedly suggesting that histone retention in sperm genes is not an all-or-none phenomenon and that a small number of histones may remain associated with genes throughout the genome. The amount of retained histones, however, was altered in many loci when PAR metabolism was impaired. To ascertain whether sperm histone association and embryonic gene expression are linked, the transcriptome of individual 2-cell embryos derived from such sperm was determined using microarrays and RNA sequencing. Strikingly, a moderate but statistically significant portion of the genes that were differentially expressed in these embryos also showed different histone retention in the corresponding gene loci in sperm of their fathers. These findings provide new evidence for the existence of a linkage between sperm histone retention and gene expression in the embryo

Competition between sigma factors for core RNA polymerase.

The switch of RNA polymerase specificity from early to late promoters of bacteriophage T4 is achieved by substitution of host sigma factor, sigma 70, with the T4 induced factor, sigma gp55. However, overproduction of sigma gp55 from an expression vector is not detrimental to Escherichia coli growth. Direct competition binding assays demonstrate that sigma 70 readily displaces sigma gp55 from RNA polymerase and thereby reverses the promoter specificity of the enzyme. The displacement also occurs with the core enzyme modified by bacteriophage T4 infection. We postulate that an antagonist of sigma 70 should be formed in T4-infected cells to aid sigma gp55 in the early/late switch

Expression of cloned rpoB gene of Escherichia coli: a genetic system for the isolation of dominant negative mutations and overproduction of defective beta subunit of RNA polymerase.

The rifampin resistance rifD18 allele of rpoB, carried on the expression plasmid pXT7 beta, is controlled by a strong bacteriophage T7 late promoter and two weak Escherichia coli promoters. Depending on the host strain, pXT7 beta specifies different levels of Rifr beta subunit, providing a system for the isolation, maintenance, and overexpression of dominant lethal alleles of rpoB. In rpoB+ hosts, pXT7 beta confers the Rifr phenotype on the Rifs host. Negative rpoB mutations in the plasmid DNA can thus be scored by screening transformants for Rifs. In an rpoB(Am) supD(Ts) host in which chromosomal rpoB expression is decreased as the temperature goes up, some of the negative plasmid-borne rpoB mutations displayed a dominant phenotype. In a host harboring inducible T7 RNA polymerase, the defective beta subunits could be overexpressed independently of the E. coli transcriptional machinery. With this system, we isolated several negative rpoB mutations induced in vitro by hydroxylamine. Seven of the mutant rpoB alleles, when overexpressed, were found to specify normal-size beta polypeptides. Two of them displayed the dominant lethal phenotype in the rpoB(Am) supD(Ts) background. We also constructed a mutation (rpoB1800) in which 24 carboxy-terminal amino acids were substituted with a random 19-amino-acid sequence. The nonfunctional rpoB1800 beta polypeptide was isolated and assembled in vitro into the core enzyme molecule

Nuclear organisation of sperm remains remarkably unaffected in the presence of defective spermatogenesis

Organisation of chromosome territories in interphase nuclei has been studied in many systems and positional alterations have been associated with disease phenotypes (e.g. laminopathies, cancer) in somatic cells. Altered nuclear organisation is also reported in developmental processes such as mammalian spermatogenesis where a "chromocentre" model is proposed with the centromeres and sex chromosomes repositioning to the nuclear centre. The purpose of this study was to test the hypothesis that alterations in nuclear organisation of human spermatozoa are associated with defects upstream in spermatogenesis (as manifest in certain infertility phenotypes). The nuclear address of (peri-) centromeric loci for 18 chromosomes (1-4, 6-12, 15-18, 20, X and Y) was assayed in 20 males using established algorithms for 3D extrapolations of 2D data. The control group comprised 10 fertile sperm donors while the test group was 10 patients with severely compromised semen parameters including high sperm aneuploidy. All loci examined in the control group adopted defined, interior positions thus providing supporting evidence for the presence of a chromocentre and interior sex chromosome territories. In the test group however there were subtle alterations in the nuclear address for certain centromeres in individual patients and, when all patient results were pooled, some different nuclear addresses were observed for chromosomes 3, 6, 12 and 18. Considering the extensive impairment of spermatogenesis in the test group (evidenced by compromised semen parameters and increased chromosome abnormalities), the observed differences in nuclear organisation for centromeric loci compared to the controls were modest. A defined pattern of nuclear reorganisation of centromeric loci in sperm heads therefore appears to be a remarkably robust process, even if spermatogenesis is severely compromised