Phosphorylation of ASF1 hampers proteasome-dependent degradation.

<p>(<b>A</b>) hASF1 and dASF1 wild type or mutant proteins were expressed in HEK293T and S2 cells respectively. Protein synthesis was blocked by cycloheximide in cells incubated with (red curves) or without (black curves) proteasome inhibitors lactacistin and MG132. ASF1 protein levels were quantified as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008328#pone-0008328-g003" target="_blank">Figure 3</a> by immunoblotting for hASF1 (upper panels) and dASF1 (lower panel) every hour from 0 to 6 h and normalized to actin. All experiments were repeated at least three times and error bars show SEM. (<b>B</b>) HA-hASF1 and HA-dASF1 proteins or GFP were expressed in HEK293T cells. Cells were either treated (+Pr.Inh.) or not (-Pr.Inh.) with proteasome inhibitors lactacistine and MG132. Whole cell extracts were incubated with anti-HA beads and pulled-down proteins were analyzed on a western blot with anti-ubiquitin antibody. High-molecular weight smear represents poly-ubiquitinated ASF1. (<b>C</b>) Degradation of endogenous dASF1 dependents only partially on proteasome pathway. Protein synthesis was blocked by cycloheximide in S2 cells incubated with (red curves) or without (black curves) proteasome inhibitors lactacistin and MG132. Samples were collected every hour from 0 to 6 h. dASF1 protein was immunoblotted and quantified as above. Data are represented as mean of three experiments +/− SEM.</p

Mutations in TLK phosphorylation sites affect ASF1 protein levels.

<p>(<b>A</b>) Mutated dASF1 proteins fused to HA-tag were co-expressed with wild-type bio-tagged dASF1 in S2 cells and revealed by immunoblotting. Mutated Serines within dASF1 protein are indicated. (<b>B</b>) Levels of mutant HA-dASF1 proteins were quantified and normalized to bio-dASF1wt protein level and compared to the same ratio obtained for HA-dASF1wt. The graph shows the mean for three experiments and error bars show standard errors of the mean (SEM). Mutated Serines are indicated (S-A mutations – blue bars, S-D – yellow bars). (<b>C, D</b>) Mutated HA-hASF1 proteins were co-expressed with GFP in HEK293T cells. Representative immunoblots are shown (<b>C</b>) and HA-hASF1 protein levels were analyzed as above (<b>D</b>) using GFP levels as a reference. The graph shows the mean for three experiments and error bars show SEM. Mutated Serines are indicated (S-A mutations – blue bars, S-D – yellow bars).</p

TLK activity is required for ASF1 stability <i>in vivo</i>.

<p>(<b>A</b>) Depletion of dTLK from Drosophila S2 cells leads to decreased dASF1 levels. S2 cells were either mock treated or incubated with dsRNA directed against dTLK. Whole-cell extracts were prepared and analyzed by Western blotting with indicated antibodies. Actin serves as a loading control. (<b>B</b>) siRNA for hTLK1 and hTLK2 affects hASF1a stability in HeLa cells. Whole-cell extracts from control or siRNA treated cells were analyzed by Western blotting with anti-hASF1a and anti-Actin antibodies. Efficiency of siRNA of hTLK1 (blue) and hTLK2 (yellow) was confirmed by RT-qPCR normalized to control siRNA (right panel).</p

Functional Dissection of the Blocking and Bypass Activities of the <i>Fab-8</i> Boundary in the <i>Drosophila</i> Bithorax Complex

<div><p>Functionally autonomous regulatory domains direct the parasegment-specific expression of the <i>Drosophila</i> Bithorax complex (BX-C) homeotic genes. Autonomy is conferred by boundary/insulator elements that separate each regulatory domain from its neighbors. For six of the nine parasegment (PS) regulatory domains in the complex, at least one boundary is located between the domain and its target homeotic gene. Consequently, BX-C boundaries must not only block adventitious interactions between neighboring regulatory domains, but also be permissive (bypass) for regulatory interactions between the domains and their gene targets. To elucidate how the BX-C boundaries combine these two contradictory activities, we have used a boundary replacement strategy. We show that a 337 bp fragment spanning the <i>Fab-8</i> boundary nuclease hypersensitive site and lacking all but 83 bp of the 625 bp <i>Fab-8</i> PTS (promoter targeting sequence) fully rescues a <i>Fab-7</i> deletion. It blocks crosstalk between the <i>iab-6</i> and <i>iab-7</i> regulatory domains, and has bypass activity that enables the two downstream domains, <i>iab-5</i> and <i>iab-6</i>, to regulate <i>Abdominal-B</i> (<i>Abd-B</i>) transcription in spite of two intervening boundary elements. <i>Fab-8</i> has two dCTCF sites and we show that they are necessary both for blocking and bypass activity. However, CTCF sites on their own are not sufficient for bypass. While multimerized dCTCF (or Su(Hw)) sites have blocking activity, they fail to support bypass. Moreover, this bypass defect is not rescued by the full length PTS. Finally, we show that orientation is critical for the proper functioning the <i>Fab-8</i> replacement. Though the inverted <i>Fab-8</i> boundary still blocks crosstalk, it disrupts the topology of the <i>Abd-B</i> regulatory domains and does not support bypass. Importantly, altering the orientation of the <i>Fab-8</i> dCTCF sites is not sufficient to disrupt bypass, indicating that orientation dependence is conferred by other factors.</p></div

The effect of the relative orientation of insulators on the interaction of <i>cis</i>-regulatory elements.

<p>In (A) and (B) the insulators pair with each other head-to-head. (A) In the insulator bypass transgene assay, a stem-loop is formed when the two insulators are in opposite orientation. This configuration is favorable for communication between regulatory elements located outside of the stem-loop. (B) When the insulators in the transgene are in the same orientation, pairing leads to the formation of a circle-loop that spatially separates regulatory elements. (C, D) The effect <i>F8</i>^<i>337</i> orientation on formation of chromatin loops. <i>Abd-B</i> regulatory region is shown at the top as green lines of different shades, with dark reflecting higher level of Abd-B expression. Insulators are shown as pentagon arrows, that indicate orientation of Fabs, with the same color as the <i>iab</i> domains they delimit. (C) In <i>F8</i>^<i>337</i>, <i>Fab-8</i> insulators are in the same orientation and head-to-head pairing between them would lead to the formation of a series of circle-loops. In this illustration the circle-loops are wound (arbitrarily) in a clockwise direction giving a right-handed helix. (D) The reversal of Fab-8³³⁷ insulator in <i>F8</i>^<i>337R</i> disrupts this helical structure and introduces two stem-loops. These loops correspond to <i>iab-6</i> and <i>iab-7</i>.</p

The phenotypic effects of <i>Fab-7</i> replacement by dCTCF or Su(Hw) binding sites, with and without PTS.

<p>In all shown homozygous mutant males A6 is transformed into A5. The phenotypic effects are the same as in the case of <i>gypsy</i> or <i>scs</i>^<i>min</i> swapping. In all shown homozygous mutant females, the transformation of A6 to A5 is evident from the appearance of a uniform trichome pattern on the entire surface of A6 tergite (dark field images).</p

The phenotypic effects of <i>Fab-7</i> replacement by <i>Fab-8</i>, with part of PTS, and by the <i>Fab-8</i> boundary inserted in the reverse orientation.

<p>All abdominal segments in <i>Fab8</i>^<i>337</i> males and females have essentially a wild type identity. The removal of the 53 bp of PTS in <i>Fab8</i>^<i>284</i> causes a weak LOF phenotype in A5 and A6. <i>Fab8</i>^<i>337R</i> induces much stronger LOF phenotypes in A6. In males, bristles appear on the A6 sternite and trichomes cover the entire surface of the A6 tergite. There is also depigmenation of the A5 tergite. In females, trichomes cover the entire A6 tergite and the pigmentation pattern resembles that of A5.</p

<i>Fab-8</i>^<i>550</i> is able to substitute for <i>Fab-7</i>.

<p>Morphology of the 5th to 8th abdominal segments (numbered) is determined by the <i>Abd-B cis</i>-regulatory regions. Wild-type (<i>wt</i>). Males: the 5th and 6th tergites are pigmented, the A6 sternite is recognizable by the absence of bristles and a specific form; A7 does not contribute to any visible cuticle structures. Trichomes are visible in the dark field and cover all the surface of A5 tergite and only a thin stripe along the anterior and ventral edges of the A6 tergite. Females: the 6th tergite is almost completely pigmented, dorsal part of the A7 tergite is depigmented, A7 sternite (ventral) has a characteristic shape with large bristles pointing towards the posterior; A8 tergite is the smallest one with no pigmentation, bristles, or trichomes. In dark field: the entire surface of the A5 tergite is evenly covered by trichomes, most of the A6 and A7 tergites is devoid of trichomes, except the anterior edges, and the ventral edge in A7. <i>F8</i>^<i>550</i> resembles wild type. <i>F8</i>^{<i>550mCTCF</i>} have mixed gain- and loss-of-function (GOF-LOF) phenotype. In males, A6 disappears completely (strong GOF transformation) but at the same time A5 acquires some features of A4 (mosaic LOF phenotype indicating a defect in the functioning of <i>iab-5</i>). Females have GOF phenotypes in A6 and A7 that are not observed in either <i>Fab-7</i> boundary deletions or in deletions that remove both the <i>Fab-7</i> boundary and the HS3 <i>iab-7</i> PRE (compare <i>F8</i>^{<i>550mCTCF</i>} females with <i>F7</i>^{<i>attP50</i>}). These include a reduction in size, an almost complete loss of pigmentation of the tergite and an abnormal pattern of bristles in the sternite. <i>F7</i>^{<i>attP50</i>} males and females have the classic GOF transformation of A6 (PS11) into A7 (PS12) seen in mutations that remove both the <i>Fab-7</i> boundary and the HS3 <i>iab-7</i> PRE.</p

Patterns of Abd-B expression in <i>CTCF</i>^<i>×4</i>, <i>Fab8</i>^<i>337</i>, <i>Fab8</i>^<i>284</i>, and <i>Fab8</i>^<i>337R</i>.

<p>Embryos were stained and marked as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006188#pgen.1006188.g003" target="_blank">Fig 3</a>. Like wild type, Abd-B expression in PS10-13 in <i>F8</i>^<i>337</i> embryos increases in a stepwise pattern from one parasegment to another. In <i>F8</i>^<i>284</i> embryos, the level of Abd-B in PS12 is elevated and close to that of PS13. In <i>F8</i>^<i>337R</i>, expression levels of Abd-B in PS13 and PS12 are nearly equal, while the Abd-B expression in PS11 is reduced. The lower panels show plot profiles of relative fluorescence intensity in the respective images from the upper panels, red lines for Abd-B and green lines for En. Parasegments are numbered from 8 to 14; approximate positions of segments are shown on the left side of the wild type (<i>wt</i>) panel and marked A4 to A8 (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006188#pgen.1006188.g001" target="_blank">Fig 1A</a> for the adult segment numbering).</p

An effect of <i>Fab-7</i> orientation on the <i>Abd-B</i> expression.

<p>Molecular maps of the <i>iab6</i>-<i>iab7</i> region, and <i>Fab-7</i> boundary replacement fragments. The <i>Fab</i>-7 insulator is represented by wide white bar on the molecular coordinate line. The part of PTS-6 is marked by dark gray. <i>Fab-7</i> has four DNase hypersensitive sites (HS*, HS1-3) shown as light gray boxes. Two variants of <i>Fab-7</i> deletions are indicated by gaps in black lines. In our experiments, <i>F7</i>^<i>858</i> and <i>F7</i>^<i>858R</i> fragments were inserted in <i>F7</i>^{<i>attP50</i>}, with a restored HS3 <i>iab-7</i> PRE in both cases. Known protein binding sites are indicated with colored ovals and rhombi. Binding factors, common with Fab-8, are shown as ovals: blue–GAF, orange–Elba/Insv. The non-common factors–as rhombi: rose–Pita, green–Zipic. Cuticles of <i>F7</i>^<i>858</i> and <i>F7</i>^<i>858R</i> males and females look essentially as wild type.</p