Gene expression analysis indicates extensive genotype-specific crosstalk between the conjugative F-plasmid and the E. coli chromosome

Background  

Plasmids are an important component of the bacterial genome, but the crosstalk between genes encoded on the chromosome and on the plasmid is still poorly understood.     

Reduced male fertility is common but highly variable in form and severity in a natural house mouse hybrid zone

Barriers to gene flow between naturally hybridizing taxa reveal the initial stages of speciation. Reduced hybrid fertility is a common feature of reproductive barriers separating recently diverged species. In house mice (Mus musculus), hybrid male sterility has been studied extensively using experimental crosses between subspecies. Here, we present the first detailed picture of hybrid male fertility in the European M. m. domesticus-M. m. musculus hybrid zone. Complete sterility appears rare or absent in natural hybrids but a large proportion of males (~30%) have sperm count or relative testis weight below the range in pure subspecies, and likely suffer reduced fertility. Comparison of a suite of traits related to fertility among subfertile males indicates reduced hybrid fertility in the contact zone is highly variable among individuals and ancestry groups in the type, number, and severity of spermatogenesis defects present. Taken together, these results suggest multiple underlying genetic incompatibilities are segregating in the hybrid zone, which likely contribute to reproductive isolation between subspecies.     

Glucocorticoids downregulate Fyn and inhibit IP3-mediated calcium signaling to promote autophagy in T lymphocytes

T cell receptor activation induces inositol 1,4,5 trisphosphate (IP₃)-mediated calcium signaling that is essential for cell metabolism and survival. Moreover, inhibitors of IP₃ or pharmacological agents that disrupt calcium homeostasis readily induce autophagy. Using a glucocorticoid-sensitive CD4/CD8 positive T cell line, we found that dexamethasone prevented both IP₃-mediated and spontaneous calcium signals within a timeframe that correlated with the induction of autophagy. We determined that this loss in IP₃-mediated calcium signaling was dependent upon the downregulation of the Src kinase Fyn at the mRNA and protein level. Because it has previously been shown that Fyn positively regulates IP₃-mediated calcium release by phosphorylating Type I IP₃ receptors (IP₃R1), we investigated the effect of glucocorticoids on IP₃R1 phosphorylation at Tyr353. Accordingly, glucocorticoid-mediated downregulation of Fyn prevented IP₃R1 phosphorylation at Tyr353. Moreover, selective knockdown of Fyn or treatment with a Src inhibitor also attenuated IP₃-mediated calcium release and induced autophagy. Collectively, these data indicate that glucocorticoids promote autophagy by inhibiting IP₃-dependent calcium signals. These findings carry important therapeutic implications given the widespread use of dexamethasone as both a chemotherapeutic and immunosuppressive agent.Key words: autophagy, calcium, Fyn, IP₃ receptor, dexamethasone     

Long microsatellite alleles in Drosophila melanogaster have a downward mutation bias and short persistence times, which cause their genome-wide underrepresentation

Microsatellites are short tandemly repeated DNA sequence motifs that are highly variable in most organisms. In contrast to mammals, long microsatellites (>15 repeats) are extremely rare in the Drosophila melanogaster genome. To investigate this paucity of long microsatellites in Drosophila, we studied 19 loci with exceptionally long microsatellite alleles. Inter- and intraspecific analysis showed that long microsatellite alleles arose in D. melanogaster only very recently. This lack of old alleles with many repeats indicated that long microsatellite alleles have short persistence times. The size distribution of microsatellite mutations in mutation-accumulation lines suggests that long alleles have a mutation bias toward a reduction in the number of repeat units. This bias causes the short persistence times of long microsatellite alleles. We propose that species-specific, size-dependent mutation spectra of microsatellite alleles may provide a general mechanism to account for the observed differences in microsatellite length between species.     

In vitro glucocorticoid sensitivity is associated with clinical glucocorticoid therapy outcome in rheumatoid arthritis

Introduction

Genetic and disease-related factors give rise to a wide spectrum of glucocorticoid (GC) sensitivity in rheumatoid arthritis (RA). In clinical practice, GC treatment is not adapted to these differences in GC sensitivity. In vitro assessment of GC sensitivity before the start of therapy could allow more individualized GC therapy. The aim of the study was to investigate the association between in vitro and in vivo GC sensitivity in RA.

Methods

Thirty-eight early and 37 established RA patients were prospectively studied. In vitro GC sensitivity was assessed with dexamethasone-induced effects on interleukin-2 (IL-2) and glucocorticoid-induced leucine zipper (GILZ) messenger RNA expression in peripheral blood mononuclear cells (PBMCs). A whole-cell dexamethasone-binding assay was used to measure number and affinity (1/K_D) of glucocorticoid receptors (GRs).In vivo GC sensitivity was determined by measuring the disease activity score (DAS) and health assessment questionnaire disability index (HAQ-DI) score before and after 2 weeks of standardized GC treatment.

Results

GR number was positively correlated with improvement in DAS. IL-2-EC₅₀ and GILZ-EC₅₀ values both had weak near-significant correlations with clinical improvement in DAS in intramuscularly treated patients only. HAQ responders had lower GILZ-EC₅₀ values and higher GR number and K_D.

Conclusions

Baseline cellular in vitro glucocorticoid sensitivity is modestly associated with in vivo improvement in DAS and HAQ-DI score after GC bridging therapy in RA. Further studies are needed to evaluate whether in vitro GC sensitivity may support the development of tailor-made GC therapy in RA.     

Localization of acetylcholine receptors and synaptic ultrastructure at nerve-muscle contacts in culture: dependence on nerve type

In cultures of xenopus myotomal muscle cells and spinal cord (SC) some of the nerve-muscle contacts exhibit a high density of acetylcholine receptors (AchRs [Anderson et al., 1977, J. Physiol. (Lond.). 268:731- 756,757-773]) and synaptic ultrastructure (Weldon and Cohen, 1979, J. Neurocytol. 8:239-259). We have examined whether similarly specialized contacts are established when the muscle cells are cultured with explants of xenopus dorsal root ganglia (DRG) or sympathetic ganglia (SG). The outgrowth from the ganglionic explants contained neuronal and non- neuronal cell processes. Although both types of processes approached within 100 A of muscle cells, synaptic ultrastructure was rarely observed at these contacts. Because patches of postsynaptic ultrastructure also develop on noncontacted muscle cells, the very few examples of contacts with such specializations probably occurred by chance. AChRs were stained with fluroscent α-bungarotoxin. More than 70 percent of the SC-contacted muscle cells exhibited a high receptor density along the path of contact. The corresponding values for DRG- and SG- contacted muscle cells were 10 and 6 percent. Similar values were obtained when the ganlionic and SC explants were cultured together in the same chamber. The few examples of high receptor density at ganglionic-muscle contacts resembled the characteristic receptor patches of noncontacted muscle cells rather than the narrow bands of high receptor density seen at SC-muscle contacts. In addition, more than 90 percent of these ganglionic- contacted muscle cells had receptor patches elsewhere, compared to less than 40 percent for the SC-contacted muscle cells. These findings indicate that the SC neurites possess a specific property which is important for the establishment of synaptically specialized contacts with muscle and that this property is lacking in the DRG and SG neurites.     

Analysis of the genomic response of human prostate cancer cells to histone deacetylase inhibitors

Histone deacetylases (HDACs) have emerged as important targets for cancer treatment. HDAC-inhibitors (HDACis) are well tolerated in patients and have been approved for the treatment of patients with cutaneous T-cell lymphoma (CTCL). To improve the clinical benefit of HDACis in solid tumors, combination strategies with HDACis could be employed. In this study, we applied Analysis of Functional Annotation (AFA) to provide a comprehensive list of genes and pathways affected upon HDACi-treatment in prostate cancer cells. This approach provides an unbiased and objective approach to high throughput data mining. By performing AFA on gene expression data from prostate cancer cell lines DU-145 (an HDACi-sensitive cell line) and PC3 (a relatively HDACi-resistant cell line) treated with HDACis valproic acid or vorinostat, we identified biological processes that are affected by HDACis and are therefore potential treatment targets for combination therapy. Our analysis revealed that HDAC-inhibition resulted among others in upregulation of major histocompatibility complex (MHC) genes and deregulation of the mitotic spindle checkpoint by downregulation of genes involved in mitosis. These findings were confirmed by AFA on publicly available data sets from HDACi-treated prostate cancer cells. In total, we analyzed 375 microarrays with HDACi treated and non-treated (control) prostate cancer cells. All results from this extensive analysis are provided as an online research source (available at the journal’s website and at http://luigimarchionni.org/HDACIs.html). By publishing this data, we aim to enhance our understanding of the cellular changes after HDAC-inhibition, and to identify novel potential combination strategies with HDACis for the treatment of prostate cancer patients.     

Epigenetic reprogramming in the porcine germ line

Background  

Epigenetic reprogramming is critical for genome regulation during germ line development. Genome-wide demethylation in mouse primordial germ cells (PGC) is a unique reprogramming event essential for erasing epigenetic memory and preventing the transmission of epimutations to the next generation. In addition to DNA demethylation, PGC are subject to a major reprogramming of histone marks, and many of these changes are concurrent with a cell cycle arrest in the G2 phase. There is limited information on how well conserved these events are in mammals. Here we report on the dynamic reprogramming of DNA methylation at CpGs of imprinted loci and DNA repeats, and the global changes in H3K27me3 and H3K9me2 in the developing germ line of the domestic pig.     

Directed targeting of chromatin to the nuclear lamina is mediated by chromatin state and A-type lamins

Nuclear organization has been implicated in regulating gene activity. Recently, large developmentally regulated regions of the genome dynamically associated with the nuclear lamina have been identified. However, little is known about how these lamina-associated domains (LADs) are directed to the nuclear lamina. We use our tagged chromosomal insertion site system to identify small sequences from borders of fibroblast-specific variable LADs that are sufficient to target these ectopic sites to the nuclear periphery. We identify YY1 (Ying-Yang1) binding sites as enriched in relocating sequences. Knockdown of YY1 or lamin A/C, but not lamin A, led to a loss of lamina association. In addition, targeted recruitment of YY1 proteins facilitated ectopic LAD formation dependent on histone H3 lysine 27 trimethylation and histone H3 lysine di- and trimethylation. Our results also reveal that endogenous loci appear to be dependent on lamin A/C, YY1, H3K27me3, and H3K9me2/3 for maintenance of lamina-proximal positioning.