A Conserved Chromatin Architecture Marks and Maintains the Restricted Germ Cell Lineage in Worms and Flies

AbstractIn C. elegans, mRNA production is initially repressed in the embryonic germline by a protein unique to C. elegans germ cells, PIE-1. PIE-1 is degraded upon the birth of the germ cell precursors, Z2 and Z3. We have identified a chromatin-based mechanism that succeeds PIE-1 repression in these cells. A subset of nucleosomal histone modifications, methylated lysine 4 on histone H3 (H3meK4) and acetylated lysine 8 on histone H4 (H4acetylK8), are globally lost and the DNA appears more condensed. This coincides with PIE-1 degradation and requires that germline identity is not disrupted. Drosophila pole cell chromatin also lacks H3meK4, indicating that a unique chromatin architecture is a conserved feature of embryonic germ cells. Regulation of the germline-specific chromatin architecture requires functional nanos activity in both organisms. These results indicate that genome-wide repression via a nanos-regulated, germ cell-specific chromatin organization is a conserved feature of germline maintenance during embryogenesis

Surface charge, fluidity, and calcium uptake by rat intestinal brush-border vesicles

AbstractBiological membrane outer surfaces are negatively charged and interact with positively charged calcium ion during calcium uptake. Positively charged polycations such as polyarginine bind to membranes with high affinity, displacing bound calcium from the membrane. We tested the effect of polyarginine on uptake of calcium by brush-border membrane vecicles and examined the responses in terms of membrane fluidity by electron paramagnetic resonance (EPR). Polyarginine inhibited the saturable component of calcium uptake by a mechanism combining inhibition characteristics of strontium (competitive) and magnesium (non-competitive). Unlike the inhibition of non-saturable calcium uptake by strontium and magnesium, polyarginine increased kD, the rate constant for non-saturable calcium uptake, by a concentration dependent mechanism. These effects of polyarginine on calcium uptake were associated with decreased membrane fluidity at the uptake temperature. These findings are consistent with a role for surface negative charge in determining both saturable and non-saturable calcium uptake. Increased membrane fluidity is associated with decreased saturable and increased non-saturable calcium uptake. Although increased fluidity might be involved in the increased kD for non-saturable uptake, the concentration-specific stimulating effect of polyarginine suggests a gating mechanism

Establishment of a Developmental Compartment Requires Interactions between Three Synergistic Cis-regulatory Modules

The subdivision of cell populations in compartments is a key event during animal development. In Drosophila, the gene apterous (ap) divides the wing imaginal disc in dorsal vs ventral cell lineages and is required for wing formation. ap function as a dorsal selector gene has been extensively studied. However, the regulation of its expression during wing development is poorly understood. In this study, we analyzed ap transcriptional regulation at the endogenous locus and identified three cis-regulatory modules (CRMs) essential for wing development. Only when the three CRMs are combined, robust ap expression is obtained. In addition, we genetically and molecularly analyzed the trans-factors that regulate these CRMs. Our results propose a three-step mechanism for the cell lineage compartment expression of ap that includes initial activation, positive autoregulation and Trithorax-mediated maintenance through separable CRMsThis study was supported by a grant from the MINECO to CE (No. BFU2012-34353) and grants from the Kantons Basel-Stadt and Basel-Land, and the Swiss National Science Foundation to MA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscrip

Selection and maintenance of sexual identity in the Drosophila germline.

Unlike sex determination in the soma, which is an autonomous process, sex determination in the germline of Drosophila has both inductive and autonomous components. In this paper, we examined how sexual identity is selected and maintained in the Drosophila germline. We show that female-specific expression of genes in the germline is dependent on a somatic signaling pathway. This signaling pathway requires the sex-non-specific transformer 2 gene but, surprisingly, does not appear to require the sex-specific genes, transformer and doublesex. Moreover, in contrast to the soma where pathway initiation and maintenance are independent processes, the somatic signaling pathway appears to function continuously from embryogenesis to the larval stages to select and sustain female germline identity. We also show that the primary target for the somatic signaling pathway in germ cells can not be the Sex-lethal gene

Macrocephaly and developmental delay caused by missense variants in RAB5C

Rab GTPases are important regulators of intracellular vesicular trafficking. RAB5C is a member of the Rab GTPase family that plays an important role in the endocytic pathway, membrane protein recycling and signaling. Here we report on 12 individuals with nine different heterozygous de novo variants in RAB5C. All but one patient with missense variants (n = 9) exhibited macrocephaly, combined with mild-to-moderate developmental delay. Patients with loss of function variants (n = 2) had an apparently more severe clinical phenotype with refractory epilepsy and intellectual disability but a normal head circumference. Four missense variants were investigated experimentally. In vitro biochemical studies revealed that all four variants were damaging, resulting in increased nucleotide exchange rate, attenuated responsivity to guanine exchange factors and heterogeneous effects on interactions with effector proteins. Studies in C. elegans confirmed that all four variants were damaging in vivo and showed defects in endocytic pathway function. The variant heterozygotes displayed phenotypes that were not observed in null heterozygotes, with two shown to be through a dominant negative mechanism. Expression of the human RAB5C variants in zebrafish embryos resulted in defective development, further underscoring the damaging effects of the RAB5C variants. Our combined bioinformatic, in vitro and in vivo experimental studies and clinical data support the association of RAB5C missense variants with a neurodevelopmental disorder characterized by macrocephaly and mild-to-moderate developmental delay through disruption of the endocytic pathway