Dynamic Chromatin Organization during Foregut Development Mediated by the Organ Selector Gene PHA-4/FoxA

Central regulators of cell fate, or selector genes, establish the identity of cells by direct regulation of large cohorts of genes. In Caenorhabditis elegans, foregut (or pharynx) identity relies on the FoxA transcription factor PHA-4, which activates different sets of target genes at various times and in diverse cellular environments. An outstanding question is how PHA-4 distinguishes between target genes for appropriate transcriptional control. We have used the Nuclear Spot Assay and GFP reporters to examine PHA-4 interactions with target promoters in living embryos and with single cell resolution. While PHA-4 was found throughout the digestive tract, binding and activation of pharyngeally expressed promoters was restricted to a subset of pharyngeal cells and excluded from the intestine. An RNAi screen of candidate nuclear factors identified emerin (emr-1) as a negative regulator of PHA-4 binding within the pharynx, but emr-1 did not modulate PHA-4 binding in the intestine. Upon promoter association, PHA-4 induced large-scale chromatin de-compaction, which, we hypothesize, may facilitate promoter access and productive transcription. Our results reveal two tiers of PHA-4 regulation. PHA-4 binding is prohibited in intestinal cells, preventing target gene expression in that organ. PHA-4 binding within the pharynx is limited by the nuclear lamina component EMR-1/emerin. The data suggest that association of PHA-4 with its targets is a regulated step that contributes to promoter selectivity during organ formation. We speculate that global re-organization of chromatin architecture upon PHA-4 binding promotes competence of pharyngeal gene transcription and, by extension, foregut development

Restricting Dosage Compensation Complex Binding to the X Chromosomes by H2A.Z/HTZ-1

Dosage compensation ensures similar levels of X-linked gene products in males (XY or XO) and females (XX), despite their different numbers of X chromosomes. In mammals, flies, and worms, dosage compensation is mediated by a specialized machinery that localizes to one or both of the X chromosomes in one sex resulting in a change in gene expression from the affected X chromosome(s). In mammals and flies, dosage compensation is associated with specific histone posttranslational modifications and replacement with variant histones. Until now, no specific histone modifications or histone variants have been implicated in Caenorhabditis elegans dosage compensation. Taking a candidate approach, we have looked at specific histone modifications and variants on the C. elegans dosage compensated X chromosomes. Using RNAi-based assays, we show that reducing levels of the histone H2A variant, H2A.Z (HTZ-1 in C. elegans), leads to partial disruption of dosage compensation. By immunofluorescence, we have observed that HTZ-1 is under-represented on the dosage compensated X chromosomes, but not on the non-dosage compensated male X chromosome. We find that reduction of HTZ-1 levels by RNA interference (RNAi) and mutation results in only a very modest change in dosage compensation complex protein levels. However, in these animals, the X chromosome–specific localization of the complex is partially disrupted, with some nuclei displaying DCC localization beyond the X chromosome territory. We propose a model in which HTZ-1, directly or indirectly, serves to restrict the dosage compensation complex to the X chromosome by acting as or regulating the activity of an autosomal repellant

Covalent Label Transfer Between Peroxisomal Importomer Components Reveals Export-Driven Import Interactions

Peroxisomes are vital metabolic organelles found in almost all eukaryotic organisms, and they rely exclusively on import of their matrix protein content from the cytosol. In vitro import of proteins into isolated peroxisomal fractions has provided a wealth of knowledge on the import process. However, the common method of protease protection garnered no information on the import of an N-terminally truncated PEX5 (PEX5C) receptor construct or peroxisomal Malate Dehydrogenase 1 (pMDH1) cargo protein into sunflower peroxisomes, owing to high degrees of protease susceptibility or resistance, respectively. Here, we present a means for analysis of in vitro import through a covalent biotin label transfer, and employ this method to the import of PEX5C. Label transfer demonstrates that PEX5C construct is monomeric in the conditions of the import assay. This technique was capable of identifying the PEX5-PEX14 interaction as the first interaction of the import process through competition experiments. Labelling of the peroxisomal protein import machinery by PEX5C demonstrated that this interaction was independent of added cargo protein, and strikingly, the interaction between PEX5C and the import machinery was shown to be ATP-dependent. These important mechanistic insights highlight the power of label transfer in studying interactions, rather than proteins, of interest, and demonstrate that this technique should be applied to future studies of peroxisomal in vitro import