Dynamic distribution of the replacement histone variant H3.3 in the mouse oocyte and preimplantation embryos

Upon fertilization, the gametes undergo a drastic reprogramming that includes changes in DNA methylation and histone modifications. Currently, it is not known whether replacement of the major histones by histone variants is also involved in these processes. Here we have examined the expression and localization of the histone variant H3.3 in early mouse embryogenesis. We show that H3.3 is present in the oocyte as a maternal factor. It is then incorporated preferentially into the male pronucleus before genome activation, pointing towards an asymmetry in histone composition between the two pronuclei. This is in line with the male pronucleus bearing transcriptional activation first. The same distribution was observed when we followed the localisation of a tagged version of H3.3. We detected H3.3 in the nuclei of mouse embryos in all of the stages analysed, from the zygote to the blastocyst stage, suggesting that the epigenetic mechanisms in the early embryo not only involve changes in histone modifications but may also include histone replacement

Binding loci of RelA-containing nuclear factor-kappaB dimers in promoter regions of PHM1-31 myometrial smooth muscle cells.

Human parturition is associated with many pro-inflammatory mediators which are regulated by the nuclear factor-kappaB (NF-κB) family of transcription factors. In the present study, we employed a ChIP-on-chip approach to define genomic loci within chromatin of PHM1-31 myometrial cells that were occupied by RelA-containing NF-κB dimers in response to a TNF stimulation of 1 h. In TNF-stimulated PHM1-31 cells, anti-RelA serum enriched 13 300 chromatin regions; importantly, 11 110 regions were also enriched by anti-RelA antibodies in the absence of TNF. DNA sequences in these regions, from both unstimulated or TNF-stimulated PHM1-31 cultures, were associated with genic regions including IκBα, COX-2, IL6RN, Jun and KCNMB3. TNF-induced binding events at a consensus κB site numbered 1667; these were represented by 112 different instances of the consensus κB motif. Of the 1667 consensus κB motif occurrences, 770 (46.2%) were identified within intronic regions. In unstimulated PHM1-31 cells, anti-RelA-serum-enriched regions were associated with sequences corresponding to open reading frames of ion channel subunit genes including CACNB3 and KCNB1. Moreover, in unstimulated cells, the consensus κB site was identified 2116 times, being defined by 103 different sequence instances of this motif. Of these 2116 consensus κB motifs, 1089 (51.5%) were identified within intronic regions. Parallel expression array analyses in PHM1-31 cultures demonstrated that TNF stimulated a >2-fold induction in 51 genes and a fold repression of >1.5 in 18 others. We identified 14 anti-RelA-serum-enriched genomic regions that correlated with 17 TNF-inducible genes, such as COX2, Egr-1, Jun, IκBα and IL6, as well as five regions associated with TNF-mediated gene repression, including Col1A2

Phenotypically distinct female castes in honey bees are defined by alternative chromatin states during larval development

The capacity of the honey bee to produce three phenotypically distinct organisms (two female castes; queens and sterile workers, and haploid male drones) from one genotype represents one of the most remarkable examples of developmental plasticity in any phylum. The queen-worker morphological and reproductive divide is environmentally controlled during post-embryonic development by differential feeding. Previous studies implicated metabolic flux acting via epigenetic regulation, in particular DNA methylation and microRNAs, in establishing distinct patterns of gene expression underlying caste-specific developmental trajectories. We produce the first genome-wide maps of chromatin structure in the honey bee at a key larval stage in which developmental canalization into queen or worker is virtually irreversible. We find extensive genome-wide differences in H3K4me3, H3K27ac, and H3K36me3, many of which correlate with caste-specific transcription. Furthermore, we identify H3K27ac as a key chromatin modification, with caste-specific regions of intronic H3K27ac directing the worker caste. These regions may harbor the first examples of caste-specific enhancer elements in the honey bee. Our results demonstrate a key role for chromatin modifications in the establishment and maintenance of caste-specific transcriptional programs in the honey bee. We show that at 96 h of larval growth, the queen-specific chromatin pattern is already established, whereas the worker determination is not, thus providing experimental support for the perceived timing of this critical point in developmental heterochrony in two types of honey bee females. In a broader context, our study provides novel data on environmentally regulated organismal plasticity and the molecular foundation of the evolutionary origins of eusociality.This work was funded by a grant to P.J.H. from the Biotechnology and Biological Sciences Research Council (BBSRC; BB/L023164/1). M.W. received funding from the William Harvey International Translational Research Academy COFUND Marie Curie Actions (WHRI-ACADEMY; PCOFUND-GA-2013-608765). Work in the Maleszka laboratory was supported by the Australian Research Council grant DP160103053

Dynamic distribution of the replacement histone variant H3.3 in the mouse oocyte and preimplantation embryos

Upon fertilization, the gametes undergo a drastic reprogramming that includes changes in DNA methylation and histone modifications. Currently, it is not known whether replacement of the major histones by histone variants is also involved in these processes. Here we have examined the expression and localization of the histone variant H3.3 in early mouse embryogenesis. We show that H3.3 is present in the oocyte as a maternal factor. It is then incorporated preferentially into the male pronucleus before genome activation, pointing towards an asymmetry in histone composition between the two pronuclei. This is in line with the male pronucleus bearing transcriptional activation first. The same distribution was observed when we followed the localisation of a tagged version of H3.3. We detected H3.3 in the nuclei of mouse embryos in all of the stages analysed, from the zygote to the blastocyst stage, suggesting that the epigenetic mechanisms in the early embryo not only involve changes in histone modifications but may also include histone replacement

Acute social isolation alters neurogenomic state in songbird forebrain

Prolonged social isolation has negative effects on brain and behavior in humans and other social organisms, but neural mechanisms leading to these effects are not understood. Here we tested the hypothesis that even brief periods of social isolation can alter gene expression and DNA methylation in higher cognitive centers of the brain, focusing on the auditory/associative forebrain of the highly social zebra finch. Using RNA sequencing, we first identified genes that individually increase or decrease expression after isolation and observed general repression of gene sets annotated for neurotrophin pathways and axonal guidance functions. We then pursued 4 genes of large effect size: EGR1 and BDNF (decreased by isolation) and FKBP5 and UTS2B (increased). By in situ hybridization, each gene responded in different cell subsets, arguing against a single cellular mechanism. To test whether effects were specific to the social component of the isolation experience, we compared gene expression in birds isolated either alone or with a single familiar partner. Partner inclusion ameliorated the effect of solo isolation on EGR1 and BDNF, but not on FKBP5 and UTS2B nor on circulating corticosterone. By bisulfite sequencing analysis of auditory forebrain DNA, isolation caused changes in methylation of a subset of differentially expressed genes, including BDNF. Thus, social isolation has rapid consequences on gene activity in a higher integrative center of the brain, triggering epigenetic mechanisms that may influence processing of ongoing experience.Peer reviewe

Ciliary entry of the kinesin-2 motor KIF17 is regulated by importin-beta2 and RanGTP

The biogenesis, maintenance, and function of primary cilia are controlled through intraflagellar transport (IFT) driven by two kinesin-2 family members, the heterotrimeric KIF3A/KIF3B/KAP complex and the homodimeric KIF17 motor1,2. How these motors and their cargoes gain access to the ciliary compartment is poorly understood. We identify a ciliary localization signal (CLS) in the KIF17 tail domain that is necessary and sufficient for ciliary targeting. Similarities between the CLS and classic nuclear localization signals (NLS) suggests that similar mechanisms regulate nuclear and ciliary import. We hypothesize that ciliary targeting of KIF17 is regulated by a Ran-GTP gradient across the ciliary base. Consistent with this, cytoplasmic expression of GTP-locked Ran(G19V) disrupts the gradient and abolishes ciliary entry of KIF17. Furthermore, KIF17 interacts with importin-β2 in a manner dependent on the CLS and inhibited by Ran-GTP. We propose that Ran plays a global role in regulating cellular compartmentalization by controlling the shuttling of cytoplasmic proteins into nuclear and ciliary compartments

Limiting distributions for explosive PAR(1) time series with strongly mixing innovation

This work deals with the limiting distribution of the least squares estimators of the coefficients a r of an explosive periodic autoregressive of order 1 (PAR(1)) time series X r = a r X r--1 +u r when the innovation {u k } is strongly mixing. More precisely {a r } is a periodic sequence of real numbers with period P \textgreater{} 0 and such that P r=1 |a r | \textgreater{} 1. The time series {u r } is periodically distributed with the same period P and satisfies the strong mixing property, so the random variables u r can be correlated

Identification of S-nitrosated mitochondrial proteins by S-nitrosothiol difference in gel electrophoresis (SNO-DIGE): implications for the regulation of mitochondrial function by reversible S-nitrosation

The S-nitrosation of mitochondrial proteins as a consequence of NO metabolism is of physiological and pathological significance. We previously developed a MitoSNO (mitochondria-targeted S-nitrosothiol) that selectively S-nitrosates mitochondrial proteins. To identify these S-nitrosated proteins, here we have developed a selective proteomic methodology, SNO-DIGE (S-nitrosothiol difference in gel electrophoresis). Protein thiols in control and MitoSNO-treated samples were blocked, then incubated with copper(II) and ascorbate to selectively reduce S-nitrosothiols. The samples were then treated with thiol-reactive Cy3 (indocarbocyanine) or Cy5 (indodicarbocyanine) fluorescent tags, mixed together and individual protein spots were resolved by 2D (two-dimensional) gel electrophoresis. Fluorescent scanning of these gels revealed S-nitrosated proteins by an increase in Cy5 red fluorescence, allowing for their identification by MS. Parallel analysis by Redox-DIGE enabled us to distinguish S-nitrosated thiol proteins from those which became oxidized due to NO metabolism. We identified 13 S-nitrosated mitochondrial proteins, and a further four that were oxidized, probably due to evanescent S-nitrosation relaxing to a reversible thiol modification. We investigated the consequences of S-nitrosation for three of the enzymes identified using SNO-DIGE (aconitase, mitochondrial aldehyde dehydrogenase and α-ketoglutarate dehydrogenase) and found that their activity was selectively and reversibly inhibited by S-nitrosation. We conclude that the reversible regulation of enzyme activity by S-nitrosation modifies enzymes central to mitochondrial metabolism, whereas identification and functional characterization of these novel targets provides mechanistic insight into the potential physiological and pathological roles played by this modification. More generally, the development of SNO-DIGE facilitates robust investigation of protein S-nitrosation across the proteome

Selective Breeding for a Behavioral Trait Changes Digit Ratio

The ratio of the length of the second digit (index finger) divided by the fourth digit (ring finger) tends to be lower in men than in women. This 2D∶4D digit ratio is often used as a proxy for prenatal androgen exposure in studies of human health and behavior. For example, 2D∶4D ratio is lower (i.e. more “masculinized”) in both men and women of greater physical fitness and/or sporting ability. Lab mice have also shown variation in 2D∶4D as a function of uterine environment, and mouse digit ratios seem also to correlate with behavioral traits, including daily activity levels. Selective breeding for increased rates of voluntary exercise (wheel running) in four lines of mice has caused correlated increases in aerobic exercise capacity, circulating corticosterone level, and predatory aggression. Here, we show that this selection regime has also increased 2D∶4D. This apparent “feminization” in mice is opposite to the relationship seen between 2D∶4D and physical fitness in human beings. The present results are difficult to reconcile with the notion that 2D∶4D is an effective proxy for prenatal androgen exposure; instead, it may more accurately reflect effects of glucocorticoids, or other factors that regulate any of many genes