Candidate regulators of MSL complex identified from the genome-wide RNAi screen.

<p>The Firefly/Renilla luciferase activity ratio and its standard deviation was determined based on quadruplicate reactions in 96-well plate format after RNAi treatment targeting the following classes of candidate genes: A) Class 1: NSL complex, PAF complex and other general transcriptional regulators; B) Class 2: Activators with potential specific DNA binding activity like CG1832.</p

Gene ontology categories that were enriched in the genome-wide RNAi screen.

<p>Gene ontology categories that were enriched in the genome-wide RNAi screen.</p

An RNAi screening system in <i>Drosophila</i> SL2 cells to identify regulators of MSL complex recruitment and function.

<p>A) Schematic of the MSL-dependent reporter system for SL2 cell-based genome-wide RNAi screening. The system includes the following three elements: 1) promoter of <i>roX2</i> gene (370 bps); 2) Firefly luciferase reporter gene; 3) <i>roX2</i> DHS (DNase I Hypersensitivity Site: 280 bp). Two additional constructs were used for secondary screening in which the promoter or CES were altered. The alternative promoter is that of the CG2690 gene and the alternative CES is the minimal 150 bp fragment of CES5C2 that were previously described <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002830#pgen.1002830-Alekseyenko1" target="_blank">[3]</a>. B) Ratio of the Firefly luciferase activity to Renilla luciferase activity for MSL complex component RNAi treatments identified from the genome-wide screen. Data shown are the average and standard deviation of two replicates from the genome-wide screening plates. Control RNAi treatment targeted the GFP gene that is not present in SL2 cells.</p

NSL complex and CG1832 enrichment at CES loci.

<p>A) Heatmaps show the average enrichment of the Chromator/Chriz and MBD-R2 components of the NSL complex surrounding MRE sequences as assayed by ChIP-chip in SL2 cells (two replicates each; data available from <a href="http://www.modENCODE.org" target="_blank">http://www.modENCODE.org</a>). The darker red color indicates greater occupancy, as measured in the log2 (ChIP/input) scale. Heatmaps are shown for sets of 150 sites containing MREs that are defined as follows: MREs in chromatin entry sites (column 1); best MREs on the X chromosome or autosome 2L (MRE motifs closest to the consensus, but not utilized, columns 2 and 4); randomly chosen MREs on the X or autosome 2L (columns 3 and 5). B) Heatmaps show the enrichment of CG1832 surrounding MREs from ChIP-seq data in SL2 cells. Enrichment of CG1832 is shown surrounding the same MREs that were examined in 4A. The log2 (ChIP/input) scale is used for binned read count; the enrichment difference is enhanced in the ChIP-seq data owing to its greater dynamic range. C) Profiles of genome-wide localization data are shown for CG1832 (red), MBD-R2 (blue), and Chromator (green), in a 30 Kb region including the <i>roX2</i> locus.</p

NSL1 and CG1832 contribute to MSL complex recruitment.

<p>A) Chromatin immunoprecipitation was conducted using an antibody specific for the MSL2 protein before and after RNAi treatment targeting the <i>paf1</i>, <i>nsl1</i> and <i>CG1832</i> genes. Four CES loci and the 3′ ends of three previously characterized MSL complex target genes were assayed <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002830#pgen.1002830-Alekseyenko1" target="_blank">[3]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002830#pgen.1002830-Larschan1" target="_blank">[5]</a>. Data shown are the average and standard deviation of at least two replicates. Normalization was conducted both to input DNA material and to the non-transcribed CG15570 gene as an internal control as previously described <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002830#pgen.1002830-Gelbart2" target="_blank">[42]</a>. B) Chromatin immunoprecipitation was conducted as described for (A) but an antibody targeting the H4K16ac histone modification (Millipore) was used instead of the MSL2 antibody. In addition to the normalization as described for (A), H4K16ac IPs were normalized to histone H3 occupancy as determined by H3 ChIP. Due to significant changes in histone H3 occupancy upon <i>paf1</i> RNAi treatment, these data were not included.</p

Beyond the Spitfire: Re-visioning Latinas in Sylvia Morales’ A Crushing Love

From Dolores del Río to Salma Hayek and from Lupe Vélez to Eva Longoria, the portrayal of Latinas in the United States has provoked debate, criticism and controversy. Since the era of silent movies, Hollywood’s depiction of the Latina has been rigidly prescribed and reductive, while Latinos and Latinas themselves have contested these problematic portrayals in more nuanced and multifaceted visions of their communities. Sylvia Morales’s documentary A Crushing Love: Chicanas, Motherhood and Activism (2009) presents a vision of activists and mothers that is radically at odds with the stereotypes of Latinas in Hollywood film and in the US media more generally.1 This chapter examines the film against the context of persistently problematic representation of Latinas in the media and the ways in which Morales’ film counters demeaning images of Chicanas through a multilayered examination of different experiences of motherhood by politically radical women

EMSA probe sequences.

<p>EMSA probe sequences.</p

A long 15-bp motif increases CLAMP binding to DNA.

<p><b>A)</b> Motifs obtained from the custom gcPBMs are shown: I) The PBM+ChIP+ motif represents the sequences that CLAMP binds both <i>in vivo</i> and <i>in vitro</i>. II) The PBM-ChIP- motif represents the sequences that are on the array but not bound by CLAMP <i>in vivo</i> or <i>in vitro</i>. The most conserved 8-bp core element is indicated by vertical dashed lines. <b>B)</b> A representation of the methodology to define the minimal CLAMP-bound motif by scanning both 5’ and 3’ of the core motif. The table shows the percentage of PBM+ChIP+ (CLAMP binding both <i>in vitro</i> and <i>in vivo</i>) sequences that overlap with PBM-ChIP- sequences (CLAMP binding neither <i>in vitro</i> nor <i>in vivo</i>) at the specified motif size. The y-axis shows the nucleotides 5’ of the 8-bp core motif and the x-axis shows the nucleotides 3’. The scale ranges from green for the maximal values to red for the minimal values. Values show the percentage of PBM+ChIP+ sequences shared with PBM-ChIP- sequences for the length window selected. A value of zero overlapping sequences represents complete separation between PBM+ChIP+ and PBM-ChIP- sequences and is obtained at 4-bp 5’ and 3-bp 3’ of the core motif. <b>C)</b> CLAMP and MSL ChIP-seq enrichments are shown for sequences containing the 8-bp core with and without additional flanking sequence matching the motif. Motif hits were found using the FIMO tool (p < E-4). All 8-bp core hits were found first and the ones overlapping with the full 15-bp motif were separated as ‘8bp + matched endogenous flank’ and the rest were grouped as ‘8bp + unmatched endogenous flank’. Since the ‘8bp + unmatched endogenous flank’ group has ~10,000 sites, the top 10,000 enrichments are shown in the CLAMP enrichment plot. Since there are ~300 CES, the top 300 enrichments are shown in the MSL enrichment plot. <b>D)</b> Binding intensities are shown for the following classes of probes: 1) probes with the optimal motif (8-bp + matched endogenous flank, red); 2) probes that have matching 8-bp core regions but the endogenous flanks do not match the motif (8-bp + unmatched endogenous flanks, blue); 3) probes that have 8-bp cores with synthetic constant flanking sequences (8-bp + unmatched synthetic flank, cyan); 4) probes that do not have the 8-bp core motif (without 8-bp, brown); 5) Intensities for C-terminal 4 zinc finger GST fusion proteins are shown for probes containing the 15-bp CLAMP motif (15–bp, 4ZF, orange). <b>E)</b> CLAMP binds to DNA containing a high affinity, 8 bp + matched flank motif in an electrophoretic mobility shift assay. Biotin-labeled DNA alone (lane 1) and DNA with MBP (lane 2) do not shift, while MBP-CLAMP forms a complex with DNA to shift the signal. This was competed away with specific (high affinity) competitor but not a non-specific competitor that contains the 8-bp core but lacks endogenous flanking sequences (8-bp + unmatched synthetic flank).</p

CLAMP binds to similar <i>in vivo</i> X-enriched binding sites in <i>D</i>. <i>miranda</i> and <i>D</i>. <i>melanogaster</i>.

<p><b>A)</b> The ratio of <i>in vitro</i> CLAMP binding site density (number of CLAMP binding sequence hits per Mb) for X versus autosomes is plotted for <i>D</i>. <i>miranda</i>, <i>D</i>. <i>melanogaster</i> and <i>A</i>. <i>gambiae</i>. Autosomes of <i>D</i>. <i>melanogaster</i> are chromosomes 2L (Muller-B), 2R (Muller-C), 3L (Muller-D), 3R (Muller-E) and 4 (Muller-F); autosomes of <i>D</i>.<i>miranda</i> are chromosomes 2 (Muller-E), 4 (Muller-B) and 5 (Muller-F); and autosomes of <i>A</i>. <i>gambiae</i> are chromosomes 2L (Muller-D), 2R (Muller-E), 3L (Muller-C), and 3R (Muller-B). <b>B)</b> The density ratios of GA-repeats on individual X-chromosome(s) vs. autosomes for different repeat lengths in the <i>D</i>. <i>melanogaster</i>, <i>D</i>. <i>miranda</i> and <i>A</i>. <i>gambiae</i> genomes are plotted. Any value above 1 indicates a higher repeat density on the X-chromosome compared to autosomes. <b>C)</b> CLAMP ChIP-seq motifs are shown for <i>D</i>. <i>melanogaster</i> and <i>D</i>. <i>Miranda</i> larval ChIP-seq data. Motifs are found using MEME-ChIP for the peak regions (peak centers +/-100bp).</p

Increasing the number of GA-dinucleotide repeats increases CLAMP occupancy.

<p><b>A)</b> A multiple linear regression to test contribution of sequence length (k-mer) and shape to overall binding. Adding dinucleotide (2mer) features to the sequence-only (1mer) model increases the performance more than adding DNA shape features, indicating the importance of dinucleotides in CLAMP-DNA recognition. Adding trinucleotide (3mer) features further increases the performance marginally. <b>B)</b> CLAMP PBM binding for GA-dinucleotide repeats of different lengths is plotted as box plot distributions. The y-axis is the PBM intensity score for each number of GA-repeats, which are plotted along the x-axis, e.g. 1 = GA, 2 = GAGA. <b>C)</b> An electrophoretic mobility shift assay to test MBP-CLAMP binding to increasing numbers of GA-repeats. The labeled probes contain GA-repeats of 4 (8-bp), 8 (16-bp), 10 (20-bp) and 15 (30-bp) centered within a 60-bp sequence. The first four lanes are reactions containing MBP control protein with DNA, and the next four are MBP-CLAMP with DNA. <b>D)</b> Input-normalized CLAMP ChIP-seq signal enrichments at GA-repeats of different lengths are given for the X-chromosome (red) and autosomes (blue) from male S2 (top) and female Kc cells (bottom). The x-axis shows the number of GA-repeats e.g. 1 = GA, 2 = GAGA.</p