HEATR2 Plays a Conserved Role in Assembly of the Ciliary Motile Apparatus

Cilia are highly conserved microtubule-based structures that perform a variety of sensory and motility functions during development and adult homeostasis. In humans, defects specifically affecting motile cilia lead to chronic airway infections, infertility and laterality defects in the genetically heterogeneous disorder Primary Ciliary Dyskinesia (PCD). Using the comparatively simple Drosophila system, in which mechanosensory neurons possess modified motile cilia, we employed a recently elucidated cilia transcriptional RFX-FOX code to identify novel PCD candidate genes. Here, we report characterization of CG31320/HEATR2, which plays a conserved critical role in forming the axonemal dynein arms required for ciliary motility in both flies and humans. Inner and outer arm dyneins are absent from axonemes of CG31320 mutant flies and from PCD individuals with a novel splice-acceptor HEATR2 mutation. Functional conservation of closely arranged RFX-FOX binding sites upstream of HEATR2 orthologues may drive higher cytoplasmic expression of HEATR2 during early motile ciliogenesis. Immunoprecipitation reveals HEATR2 interacts with DNAI2, but not HSP70 or HSP90, distinguishing it from the client/chaperone functions described for other cytoplasmic proteins required for dynein arm assembly such as DNAAF1-4. These data implicate CG31320/HEATR2 in a growing intracellular pre-assembly and transport network that is necessary to deliver functional dynein machinery to the ciliary compartment for integration into the motile axoneme

The Transcription Factor Rfx3 Regulates β-Cell Differentiation, Function, and Glucokinase Expression

OBJECTIVE: Pancreatic islets of perinatal mice lacking the transcription factor Rfx3 exhibit a marked reduction in insulin-producing beta-cells. The objective of this work was to unravel the cellular and molecular mechanisms underlying this deficiency. RESEARCH DESIGN AND METHODS: Immunofluorescence studies and quantitative RT-PCR experiments were used to study the emergence of insulin-positive cells, the expression of transcription factors implicated in the differentiation of beta-cells from endocrine progenitors, and the expression of mature beta-cell markers during development in Rfx3(-/-) and pancreas-specific Rfx3-knockout mice. RNA interference experiments were performed to document the consequences of downregulating Rfx3 expression in Min6 beta-cells. Quantitative chromatin immunoprecipitation (ChIP), ChIP sequencing, and bandshift experiments were used to identify Rfx3 target genes. RESULTS: Reduced development of insulin-positive cells in Rfx3(-/-) mice was not due to deficiencies in endocrine progenitors or beta-lineage specification, but reflected the accumulation of insulin-positive beta-cell precursors and defective beta-cells exhibiting reduced insulin, Glut-2, and Gck expression. Similar incompletely differentiated beta-cells developed in pancreas-specific Rfx3-deficient embryos. Defective beta-cells lacking Glut-2 and Gck expression dominate in Rfx3-deficent adults, leading to glucose intolerance. Attenuated Glut-2 and glucokinase expression, and impaired glucose-stimulated insulin secretion, were also induced by RNA interference-mediated inhibition of Rfx3 expression in Min6 cells. Finally, Rfx3 was found to bind in Min6 cells and human islets to two well-known regulatory sequences, Pal-1 and Pal-2, in the neuroendocrine promoter of the glucokinase gene. CONCLUSIONS: Our results show that Rfx3 is required for the differentiation and function of mature beta-cells and regulates the beta-cell promoter of the glucokinase gene