Spatial organization of transcribing loci during early genome activation in Drosophila.

The early Drosophila embryo provides unique experimental advantages for addressing fundamental questions of gene regulation at multiple levels of organization, from individual gene loci to the entire genome. Using 1.5-h-old Drosophila embryos undergoing the first wave of genome activation,1 we detected ∼110 discrete "speckles" of RNA polymerase II (RNA Pol II) per nucleus, two of which were larger and localized to the histone locus bodies (HLBs).2,3 In the absence of the primary driver of Drosophila genome activation, the pioneer factor Zelda (Zld),1,4,5 70% fewer speckles were present; however, the HLBs tended to be larger than wild-type (WT) HLBs, indicating that RNA Pol II accumulates at the HLBs in the absence of robust early-gene transcription. We observed a uniform distribution of distances between active genes in the nuclei of both WT and zld mutant embryos, indicating that early co-regulated genes do not cluster into nuclear sub-domains. However, in instances whereby transcribing genes did come into close 3D proximity (within 400 nm), they were found to have distinct RNA Pol II speckles. In contrast to the emerging model whereby active genes are clustered to facilitate co-regulation and sharing of transcriptional resources, our data support an "individualist" model of gene control at early genome activation in Drosophila. This model is in contrast to a "collectivist" model, where active genes are spatially clustered and share transcriptional resources, motivating rigorous tests of both models in other experimental systems.

Intersectin links WNK kinases to endocytosis of ROMK1.

With-no-lysine (WNK) kinases are a novel family of protein kinases characterized by an atypical placement of the catalytic lysine. Mutations of 2 family members, WNK1 and WNK4, cause pseudohypoaldosteronism type 2 (PHA2), an autosomal-dominant disease characterized by hypertension and hyperkalemia. WNK1 and WNK4 stimulate clathrin-dependent endocytosis of renal outer medullar potassium 1 (ROMK1), and PHA2-causing mutations of WNK4 increase the endocytosis. How WNKs stimulate endocytosis of ROMK1 and how mutations of WNK4 increase the endocytosis are unknown. Intersectin (ITSN) is a multimodular endocytic scaffold protein. Here we show that WNK1 and WNK4 interacted with ITSN and that the interactions were crucial for stimulation of endocytosis of ROMK1 by WNKs. The stimulation of endocytosis of ROMK1 by WNK1 and WNK4 required specific proline-rich motifs of WNKs, but did not require their kinase activity. WNK4 interacted with ROMK1 as well as with ITSN. Disease-causing WNK4 mutations enhanced interactions of WNK4 with ITSN and ROMK1, leading to increased endocytosis of ROMK1. These results provide a molecular mechanism for stimulation of endocytosis of ROMK1 by WNK kinases.

The Drosophila Pioneer Factor Zelda Modulates the Nuclear Microenvironment of a Dorsal Target Enhancer to Potentiate Transcriptional Output.

Connecting the developmental patterning of tissues to the mechanistic control of RNA polymerase II remains a long-term goal of developmental biology. Many key elements have been identified in the establishment of spatial-temporal control of transcription in the early Drosophila embryo, a model system for transcriptional regulation. The dorsal-ventral axis of the Drosophila embryo is determined by the graded distribution of Dorsal (Dl), a homolog of the nuclear factor κB (NF-κB) family of transcriptional activators found in humans [1, 2]. A second maternally deposited factor, Zelda (Zld), is uniformly distributed in the embryo and is thought to act as a pioneer factor, increasing enhancer accessibility for transcription factors, such as Dl [3-9]. Here, we utilized the MS2 live imaging system to evaluate the expression of the Dl target gene short gastrulation (sog) to better understand how a pioneer factor affects the kinetic parameters of transcription. Our experiments indicate that Zld modifies probability of activation, the timing of this activation, and the rate at which transcription occurs. Our results further show that this effective rate increase is due to an increased accumulation of Dl at the site of transcription, suggesting that transcription factor "hubs" induced by Zld [10] functionally regulate transcription.

phyllopod is a target gene of proneural proteins in Drosophila external sensory organ development.

Proneural basic helix-loop-helix (bHLH) proteins initiate neurogenesis in both vertebrates and invertebrates. The Drosophila Achaete (Ac) and Scute (Sc) proteins are among the first identified members of the large bHLH proneural protein family. phyllopod (phyl), encoding an ubiquitin ligase adaptor, is required for ac- and sc-dependent external sensory (ES) organ development. Expression of phyl is directly activated by Ac and Sc. Forced expression of phyl rescues ES organ formation in ac and sc double mutants. phyl and senseless, encoding a Zn-finger transcriptional factor, depend on each other in ES organ development. Our results provide the first example that bHLH proneural proteins promote neurogenesis through regulation of protein degradation.