This result suggests that PHA-4 is a critical contributor to large-scale decompaction of especially at early developmental stages. PHA-4::GFP signal (Arrows; Left image) compared to a transgenic line expressing PHA-4::GFP without any target promoter (Right image). (D) PHA-4 binding is maintained on mitotic chromosomes (Arrows) (E) The diameter of pharyngeal nuclei at different developmental stages.(8.08 MB TIF) pgen.1001060.s005.tif (7.7M) GUID:?B297EA92-AA75-4D09-883F-B05EB59D4C48 Figure S6: RNAi reduces the expression of EMR-1 in all cells. (A) EMR-1 antibody stain reveals its position at the nuclear periphery in a nuclear spot assay transgenic line (B) EMR-1 signal is lost after RNAi. A secondary antibody against LacI was used as a positive control for antibody staining (LacI alone shown in the inset).(5.57 MB TIF) pgen.1001060.s006.tif (5.3M) GUID:?69490537-E269-4077-89BF-B97D0D12D9F3 Figure S7: Comparison of area measurements versus volume measurements for array size. (A) Area or (B) Volume of pseudo-chromosomes in the pharynx were measured at the comma and 1.5Fold stage in transgenic lines carrying either a WT promoter or a promoter. Three embryos per stage were analyzed. Each dot on the plot represents a pseudo-chromosome.(7.06 MB TIF) pgen.1001060.s007.tif (6.7M) GUID:?263CB07D-E4C7-448B-8E50-AFB1A96734E5 Table S1: reporters are activated at the bean stage. (A) GFP expression assayed in two transgenic lines. (A) is a line carrying a transcriptional fusion of 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 GSK726701A 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 GSK726701A 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 GSK726701A pharyngeal gene transcription and, by extension, foregut development. Author Summary Central regulators of cell fate establish the identity of cells by direct regulation of large cohorts of genes. In plays a broad role in the development and physiology of GSK726701A the digestive tract. PHA-4 establishes the diverse cell types of the pharynx during early embryogenesis, and drives differentiation and morphogenesis at later stages C. After birth, PHA-4 is required for growth and gonadogenesis in larvae , C and promotes longevity in adults , . The targets of PHA-4 are likely distinct in different tissues and at different developmental stages. For example, numerous PHA-4 target genes have been identified within the pharynx, but most of these are not active in the intestine or gonad , , . Recent chromatin immunoprecipitation data with tagged PHA-4 suggest different genes are bound by PHA-4 at different developmental stages . How is appropriate regulation of PHA-4 target genes achieved? One mechanism is combinatorial control by PHA-4 with other transcription factors. A single PHA-4 binding site is not sufficient for transcriptional activation, and most foregut promoters carry four or more cis-regulatory elements that contribute towards appropriate spatial and temporal expression , , C. In addition, DNA binding affinity of PHA-4 for target genes modulates the timing of activation , . GSK726701A High affinity sites promote earlier transcriptional onset compared to lower affinity sites, within the context of the intact cis regulatory region . These studies suggest that binding affinity, feed-forward loops, positive feedback and combinatorial control, are necessary to achieve accurate temporal gene expression. However, it is still largely unknown how spatial regulation is accomplished. For example, why are pharyngeal genes active in the pharynx but not in Rabbit polyclonal to ANKRD49 the intestine, despite the widespread expression of PHA-4 in both organs? Studies have implicated the nuclear periphery for modulation of gene transcription. Active and inducible genes are recruited to nuclear pores C. Conversely, nuclear lamins and their associated proteins have been associated with transcriptional repression and chromatin organization C. Inactive genes are often positioned at.