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

Phenotypes from initiator mutants.

<p>Genotypes are as follows: A.–C. <i>iab-6¹</i>, D.–F. <i>iab-6⁴</i> and G.–I. <i>iab-6⁸</i>. A., D. and G. show adult male cuticles. B., E. and H. show pseudo-darkfield views of the fifth and sixth tergites to visualize the trichome patterns. C., F. and I. show the Abd-B staining pattern in the embryonic nerve cord. In wild-type flies, A5/PS10 differs from A6/PS11 based on the sternite shape, the bristles present on the A5 sternite, the trichome pattern on the fifth and sixth tergites, and the Abd-B staining pattern in the CNS (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1001260#pgen-1001260-g003" target="_blank">Figure 3</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1001260#pgen-1001260-g004" target="_blank">Figure 4</a>). The <i>iab-6¹</i> and <i>iab-6⁴</i> show transformations of A6 to A5 for all phenotypes monitored. Meanwhile <i>iab-6⁸</i> shows only a partial transformation of A6 to A5 as seen by the sternite shape and trichome pattern on A6, which remain A6-like.</p

<i>iab-5,6^CI</i> phenotype and rescue.

<p>A. A wild-type adult male cuticle with A4-A6 labeled. Segment A5 differs from A6 based on the sternite shape and the bristles present on the A5 sternite. For reference, the A6 tergite is indicated by a red arrowhead and the A6 sternite is indicated by a red arrow. B. A wild-type embryonic nerve cord (anterior towards the top) stained with an antibody to <i>Abd-B</i> (brown). Notice the step gradient of <i>Abd-B</i> expression increasing in each parasegment towards the posterior. C. An adult male cuticle of a fly homozygous for the <i>iab-5,6^CI</i> chromosome with A5 and A6 transformed towards A4 (notice the A4-like pigmentation on the tergites and the bristled sternites). D. The embryonic nerve cord of homozygous <i>iab-5,6^CI</i> embryos shows only a transformation of A6 into A5, as seen by the repetition of PS10/A5-like Abd-B levels in PS11/A6, indicating that the inactivation of <i>iab-5</i> is incomplete and not seen in the embryo. E. An adult male cuticle from a fly homozygous for the <i>iab-5,6^rescue</i> chromosome, where the entire 19.3 kb area deleted in <i>iab-5,6^CI</i> is reintegrated into <i>iab-5,6^CI</i>, looks completely wild type. F. The complete rescue is confirmed by the wild-type pattern of <i>Abd-B</i> in the embryonic ventral nerve cord.</p

<i>Fab-6</i> boundary mutations.

<p>The genotypes of the adult male cuticles of A. <i>Fab-6²</i>, and B. <i>Fab-6³.</i> C. (wild type) and D. (<i>Fab-6³</i>) are embryonic nerve cords stained for Abd-B protein. Notice the increased level of Abd-B in PS10 in mutants (D.) relative to wild-type (C.).</p

Oligos used to generate the deletions.

<p>Bold sequences correspond to the FRT-kanamycin-FRT sequences used to prime the amplification of the FRT-kanamycin-FRT cassette. Regular characters correspond to the homology regions used to generate the deletions by recombineering. P1–P7 correspond to the oligos used to generate the proximal breakpoint of the deletions (relative to the <i>Abd-B</i> promoter), while D1–D8 correspond to the oligos used to generate the distal breakpoint.</p

Phenotypes from initiator mutants.

<p>Genotypes are as follows: A. and D. <i>iab-6⁴</i>. B. and E. wild type. C. and F. <i>Fab-6^IAB5</i>. A.–C. Show the ventral sternite cuticles made from adult males, homozygous for the genotype indicated above. Notice that A5 differs from A6 based on the sternite shape and the bristles present on the A5 sternite. The opposite homeotic transformations are highlighted by the direction of the arrows on the left and the right of the cuticles. D.–F. Show ventral nerve chords made from homozygous embryos of the genotypes indicated above. Parasegment borders are marked to the left.</p

Synopsis of the <i>Abd-B</i> locus of the BX-C and diagram of the mutations created for this study.

<p>A. Synopsis of the <i>Abd-B</i> locus of the BX-C. Diagram of the <i>Abd-B</i> gene and its 3′<i>cis-</i>regulatory region. The horizontal line represents the DNA sequence of the BX-C (see scale on top left). The <i>Abd-B</i> expression pattern in the central nervous system of a 10 hours embryo is shown above the DNA line. In parasegment 10 (PS10) <i>Abd-B</i> is present in a few nuclei at a relatively low level. This PS10-specific expression pattern is controlled by the <i>iab-5</i> regulatory domain located 55 kb downstream from the <i>Abd-B</i> promoter. In PS11, PS12 and PS13, <i>Abd-B</i> is present in progressively more nuclei and at higher levels. These patterns are controlled by the <i>iab-6</i>, <i>iab-7</i> and <i>iab-8</i> regulatory domains, respectively. Each regulatory domain functions autonomously from its neighbors due to the presence of the boundaries that flank them (red ovals). B. Diagram of the mutations created for this study. The top line shows the DNA coordinates of <i>iab-6</i>, according to the <i>Drosophila</i> Genome Project. Below this line, and to approximate scale, are the locations of the various elements isolated from the BX-C including the IAB5 initiator<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1001260#pgen.1001260-Busturia1" target="_blank">[12]</a>, DNase hypersentive site 1 (HS1/<i>Fab-6</i> including the CTCF binding sites) and 2 (HS2/PRE) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1001260#pgen.1001260-PerezLluch1" target="_blank">[43]</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1001260#pgen.1001260-Holohan1" target="_blank">[44]</a>, the 2.8 kb <i>iab-6</i> initiator fragment <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1001260#pgen.1001260-Mihaly1" target="_blank">[22]</a>, the minimal initiator fragment and the <i>Fab-7</i> boundary <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1001260#pgen.1001260-Hagstrom1" target="_blank">[14]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1001260#pgen.1001260-Gyurkovics1" target="_blank">[30]</a>. Below this line are the DNAs reintegrated to make the mutations. The various <i>iab-6</i> alleles are indicated as solid bars, with gaps indicating the areas deleted. These bars are color coded such that blue bars indicate mutants that show no cuticle or CNS phenotypes at 25°C, red bars indicate mutants with <i>Fab-6</i>-type phenotypes, turquoise bars indicate mutants with <i>iab-5,6</i> phenotypes, and green bars indicate mutants with <i>iab-6</i> phenotypes.</p

PLEKHA5, PLEKHA6, and PLEKHA7 bind to PDZD11 to target the Menkes ATPase ATP7A to the cell periphery and regulate copper homeostasis

The WW-PLEKHA proteins PLEKHA5, PLEKHA6, and PLEKHA7 coordinate together with PDZD11 the anterograde traffic of the copper pump ATP7A from the trans-Golgi network to the cell periphery. WW-PLEKHAs promote PDZD11 interaction with the C-terminus of ATP7A and are required for maintaining low intracellular copper levels when cells face elevated copper.</p

Cingulin and paracingulin tether myosins-2 to junctions to mechanoregulate the plasma membrane

The mechanisms that regulate the spatial sorting of nonmuscle myosins-2 (NM2) isoforms and couple them mechanically to the plasma membrane are unclear. Here we show that the cytoplasmic junctional proteins cingulin (CGN) and paracingulin (CGNL1) interact directly with NM2s through their C-terminal coiled-coil sequences. CGN binds strongly to NM2B, and CGNL1 to NM2A and NM2B. Knockout (KO), exogenous expression, and rescue experiments with WT and mutant proteins show that the NM2-binding region of CGN is required for the junctional accumulation of NM2B, ZO-1, ZO-3, and phalloidin-labeled actin filaments, and for the maintenance of tight junction membrane tortuosity and apical membrane stiffness. CGNL1 expression promotes the junctional accumulation of both NM2A and NM2B and its KO results in myosin-dependent fragmentation of adherens junction complexes. These results reveal a mechanism for the junctional localization of NM2A and NM2B and indicate that, by binding to NM2s, CGN and CGNL1 mechanically couple the actomyosin cytoskeleton to junctional protein complexes to mechanoregulate the plasma membrane.</p