Initiator Elements Function to Determine the Activity State of BX-C Enhancers

A >300 kb cis-regulatory region is required for the proper expression of the three bithorax complex (BX-C) homeotic genes. Based on genetic and transgenic analysis, a model has been proposed in which the numerous BX-C cis-regulatory elements are spatially restricted through the activation or repression of parasegment-specific chromatin domains. Particular early embryonic enhancers, called initiators, have been proposed to control this complex process. Here, in order to better understand the process of domain activation, we have undertaken a systematic in situ dissection of the iab-6 cis-regulatory domain using a new method, called InSIRT. Using this method, we create and genetically characterize mutations affecting iab-6 function, including mutations specifically modifying the iab-6 initiator. Through our mutagenesis of the iab-6 initiator, we provide strong evidence that initiators function not to directly control homeotic gene expression but rather as domain control centers to determine the activity state of the enhancers and silencers within a cis-regulatory domain

Paracingulin recruits CAMSAP3 to tight junctions and regulates microtubule and polarized epithelial cell organization

Paracingulin (CGNL1) is recruited to tight junctions (TJs) by ZO-1 and to adherens junctions (AJs) by PLEKHA7. PLEKHA7 has been reported to bind to the microtubule minus-end-binding protein CAMSAP3, to tether microtubules to the AJs. Here, we show that knockout (KO) of CGNL1, but not of PLEKHA7, results in the loss of junctional CAMSAP3 and its redistribution into a cytoplasmic pool both in cultured epithelial cells in vitro and mouse intestinal epithelium in vivo. In agreement, GST pulldown analyses show that CGNL1, but not PLEKHA7, interacts strongly with CAMSAP3, and the interaction is mediated by their respective coiled-coil regions. Ultrastructure expansion microscopy shows that CAMSAP3-capped microtubules are tethered to junctions by the ZO-1-associated pool of CGNL1. The KO of CGNL1 results in disorganized cytoplasmic microtubules and irregular nuclei alignment in mouse intestinal epithelial cells, altered cyst morphogenesis in cultured kidney epithelial cells, and disrupted planar apical microtubules in mammary epithelial cells. Together, these results uncover new functions of CGNL1 in recruiting CAMSAP3 to junctions and regulating microtubule cytoskeleton organization and epithelial cell architecture.</p

InSIRT.

<p>A. Step one: Homologous Recombination. The original “ends-out” donor vector (pW25) was modified to contain an <i>attP</i> insertion site and a removable <i>yellow</i> reporter gene. Using the yellow reporter, homologous recombination events could be identified by screening for flies with yellow expression in the A5 and A6 segments (a consequence of having <i>yellow</i> inserted in the <i>iab-5</i> domain). The yellow reporter could then be removed to leave only the <i>attP</i> site and a single <i>loxP</i> recombination site (white triangle) in place of <i>iab-6</i>. B. Step two: Reintegration. Plasmids containing a 288 bp <i>attB</i> site, a single <i>loxP</i> site, a yellow reporter and a version of the 19.3 kb fragment were injected into <i>iab-5,6^CI</i> embryos expressing a maternally supplied φC31 integrase <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1001260#pgen.1001260-Bischof1" target="_blank">[36]</a>. Integration events were isolated based on yellow gene expression, then crossed to the Cre recombinase to remove the yellow gene and all vector backbone sequence.</p