ࡱ > a d ` y dn bjbj ;x { { 8 + z z 8 : * @ @ @ $ ! u$ n 9 @ @ 7 b * @ @ l @ ;(T)( E H $ $ $ 0 $ z : SUPPLEMENTARY METHODS
TCGA Analysis: CAP-D3, CAP-G2, and SMC4 mRNA expression was assessed via TCGA
(The Cancer Genome Atlas) low grade glioma (LGG) and glioblastoma (GBM) data sets with the use of UCSC xena (http//xena.ucsc,edu/). Two expression groups were created for each gene based on median expression and then log rank rest was used to analyze the survival differences that existed between the groups using GraphPad.
Western Blotting: Twenty larvae ubiquitously overexpressing GFP or GFP-dCAP-D3 were homogenized in RIPA lysis buffer (250mM NaCl, 50mM Tris, pH 7.5, 1mM EDTA, 0.1% Triton, 10% glycerol, 1mM DTT, 1X protease inhibitor cocktail) and incubated on ice for 30 minutes. The lysate was cleared by centrifugation and protein quantified by Bradford assay (Bio-Rad). Samples were boiled for 5 minutes in Lamaelli buffer and 50g of lysate was loaded on a denaturing SDS-polyacrylamide gel. Membranes were blocked and blotted with 5% milk/0.1%PBS-Tween with antibodies at the following concentrations (dCAP-D3ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1101/gad.1631508", "ISBN" : "0890-9369 (Print)\\r0890-9369 (Linking)", "ISSN" : "08909369", "PMID" : "18367646", "abstract" : "The Drosophila retinoblastoma family of proteins (RBF1 and RBF2) and their mammalian homologs (pRB, p130, and p107) are best known for their regulation of the G1/S transition via the repression of E2F-dependent transcription. However, RB family members also possess additional functions. Here, we report that rbf1 mutant larvae have extensive defects in chromatin condensation during mitosis. We describe a novel interaction between RBF1 and dCAP-D3, a non-SMC component of the Condensin II complex that links RBF1 to the regulation of chromosome structure. RBF1 physically interacts with dCAP-D3, RBF1 and dCAP-D3 partially colocalize on polytene chromosomes, and RBF1 is required for efficient association of dCAP-D3 with chromatin. dCap-D3 mutants also exhibit chromatin condensation defects, and mutant alleles of dCap-D3 suppress cellular and developmental phenotypes induced by the overexpression of RBF1. Interestingly, this interaction is conserved between flies and humans. The re-expression of pRB into a pRB-deficient human tumor cell line promotes chromatin association of hCAP-D3 in a manner that depends on the LXCXE-binding cleft of pRB. These results uncover an unexpected link between pRB/RBF1 and chromatin condensation, providing a mechanism by which the functional inactivation of RB family members in human tumor cells may contribute to genome instability.", "author" : [ { "dropping-particle" : "", "family" : "Longworth", "given" : "Michelle S.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Herr", "given" : "Anabel", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ji", "given" : "Jun Yuan", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Dyson", "given" : "Nicholas J.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "Genes and Development", "id" : "ITEM-1", "issued" : { "date-parts" : [ [ "2008" ] ] }, "page" : "1011-1024", "title" : "RBF1 promotes chromatin condensation through a conserved interaction with the Condensin II protein dCAP-D3", "type" : "article-journal", "volume" : "22" }, "uris" : [ "http://www.mendeley.com/documents/?uuid=37f8ec05-9cf6-41c6-bfe8-211e55abe982" ] } ], "mendeley" : { "formattedCitation" : "(Longworth et al. 2008)", "plainTextFormattedCitation" : "(Longworth et al. 2008)", "previouslyFormattedCitation" : "(Longworth et al. 2008)" }, "properties" : { "noteIndex" : 0 }, "schema" : "https://github.com/citation-style-language/schema/raw/master/csl-citation.json" }(Longworth et al. 2008) 1:1,000, GFP (abcam6556) 1:5,000). Blot images were developed with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher) and images captured on a Protec X-Ray Film Processor.
Eclosion Rates: An equal number of female virgins were crossed to an equal number of males of the same genotype and allowed to mate for 24 hours, at which point flies were emptied out of the vials. Crosses were monitored daily and the total number of larvae was noted for each genotype. The number of eclosed flies were tallied each day, and reported as a combined percentage hatched relative to the total number of identified larvae.
Body Weight Analysis: Fifty, 0-2 day old males were collected and weighed in a previously weighed eppendorf tube. The weight of the flies was calculated by subtracting the weight of the empty eppendorf. A total of five replicates were performed for each genotype.
Drosophila Cell Culture: S2 cells were obtained from the Drosophila Genomics Resource Center (S2-DRSC). Cells were cultured in Shields and Sangs M3 Insect Medium (Sigma S3652) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. pUASt-GFP-dCAP-D3 and pUASt-GFP constructs were made as previously described in ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "DOI" : "10.1371/journal.pgen.1002618", "ISSN" : "15537390", "PMID" : "22496667", "abstract" : "Previously, we discovered a conserved interaction between RB proteins and the Condensin II protein CAP-D3 that is important for ensuring uniform chromatin condensation during mitotic prophase. The Drosophila melanogaster homologs RBF1 and dCAP-D3 co-localize on non-dividing polytene chromatin, suggesting the existence of a shared, non-mitotic role for these two proteins. Here, we show that the absence of RBF1 and dCAP-D3 alters the expression of many of the same genes in larvae and adult flies. Strikingly, most of the genes affected by the loss of RBF1 and dCAP-D3 are not classic cell cycle genes but are developmentally regulated genes with tissue-specific functions and these genes tend to be located in gene clusters. Our data reveal that RBF1 and dCAP-D3 are needed in fat body cells to activate transcription of clusters of antimicrobial peptide (AMP) genes. AMPs are important for innate immunity, and loss of either dCAP-D3 or RBF1 regulation results in a decrease in the ability to clear bacteria. Interestingly, in the adult fat body, RBF1 and dCAP-D3 bind to regions flanking an AMP gene cluster both prior to and following bacterial infection. These results describe a novel, non-mitotic role for the RBF1 and dCAP-D3 proteins in activation of the Drosophila immune system and suggest dCAP-D3 has an important role at specific subsets of RBF1-dependent genes.", "author" : [ { "dropping-particle" : "", "family" : "Longworth", "given" : "Michelle S.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Walker", "given" : "James A.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Anderssen", "given" : "Endre", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Moon", "given" : "Nam Sung", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Gladden", "given" : "Andrew", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Heck", "given" : "Margarete M S", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Ramaswamy", "given" : "Sridhar", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Dyson", "given" : "Nicholas J.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "PLoS Genetics", "id" : "ITEM-1", "issued" : { "date-parts" : [ [ "2012" ] ] }, "title" : "A shared role for RBF1 and dCAP-D3 in the regulation of transcription with consequences for innate immunity", "type" : "article-journal", "volume" : "8" }, "uris" : [ "http://www.mendeley.com/documents/?uuid=c6aee6de-ed06-49c5-be18-33ac9cdc286a" ] } ], "mendeley" : { "formattedCitation" : "(Longworth et al. 2012)", "plainTextFormattedCitation" : "(Longworth et al. 2012)" }, "properties" : { "noteIndex" : 0 }, "schema" : "https://github.com/citation-style-language/schema/raw/master/csl-citation.json" }(Longworth et al. 2012) and at https://emb.carnegiescience.edu/drosophila-gateway-vector-collection using the pAGW construct. Cells were transfected with 2g of DNA using Fugene 6 (Promega). 48 hours after transfection, cells were fixed, stained with DAPI, and imaged using a Bio-Rad MRC 1000 laser scanning confocal microscope.
Nuclear Area Measurements: All images were acquired at the same magnification. Nuclear area was measured using ImageJ similar to methods described in ADDIN CSL_CITATION { "citationItems" : [ { "id" : "ITEM-1", "itemData" : { "PMID" : "22956908", "abstract" : "The eukaryotic nucleus is both spatially and functionally partitioned. This organization contributes to the maintenance, expression, and transmission of genetic information. Though our ability to probe the physical structure of the genome within the nucleus has improved substantially in recent years, relatively little is known about the factors that regulate its organization or the mechanisms through which specific organizational states are achieved. Here, we show that Drosophila melanogaster Condensin II induces axial compaction of interphase chromosomes, globally disrupts interchromosomal interactions, and promotes the dispersal of peri-centric heterochromatin. These Condensin II activities compartmentalize the nucleus into discrete chromosome territories and indicate commonalities in the mechanisms that regulate the spatial structure of the genome during mitosis and interphase.", "author" : [ { "dropping-particle" : "", "family" : "Bauer", "given" : "Christopher R.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Hartl", "given" : "Tom A.", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" }, { "dropping-particle" : "", "family" : "Bosco", "given" : "Giovanni", "non-dropping-particle" : "", "parse-names" : false, "suffix" : "" } ], "container-title" : "PLoS Genetics", "id" : "ITEM-1", "issue" : "8", "issued" : { "date-parts" : [ [ "2012" ] ] }, "title" : "Condensin II Promotes the Formation of Chromosome Territories by Inducing Axial Compaction of Polyploid Interphase Chromosomes", "type" : "article-journal", "volume" : "8" }, "uris" : [ "http://www.mendeley.com/documents/?uuid=90b49c56-b24e-4c7f-bc95-24f784489f92" ] } ], "mendeley" : { "formattedCitation" : "(Bauer et al. 2012)", "plainTextFormattedCitation" : "(Bauer et al. 2012)", "previouslyFormattedCitation" : "(Bauer et al. 2012)" }, "properties" : { "noteIndex" : 0 }, "schema" : "https://github.com/citation-style-language/schema/raw/master/csl-citation.json" }(Bauer et al. 2012). Briefly, a single z-slice was selected where nuclear area appeared to be at a maximum. A circle was superimposed over the nucleus and its area measured. This was performed separately with both the DAPI and lamin channels.
SUPPLEMENTARY SUPPLEMENTARY FIGURE FILE LEGENDS
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Figure S1: dCAP-D3 is overexpressed in whole third instar larvae. Immunobloting with antibodies against dCAP-D3 (top) and GFP (bottom) on GFP-immunoprecipitated lysate from larvae ubiquitously expressing GFP ( - T u b u l i n - G A L 4 > p U A S t - G F P ) o r G F P - d C A P - D 3 ( - T u b u l i n - G A L 4 > p U A S t - G F P - d C A P - D 3 ) .
F i g u r e S 2 d C A P - D 3 a g g r e g a t e f o r m a t i o n . i s n o t a n a r t i f a c t o f t h e G F P - t a g F L A G - H A o r F L A G - H A - d C A P - D 3 w a s o v e r e x p r e s s e d i n A ) s a l i v a r y g l a n d s u s i n g S G S - G A L 4 o r i n B ) w i n g d i s cs using Nubbin-GAL4. Immunostaining for HA is shown in gray and DAPI to label nuclei is shown in blue.
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Figure S3 dCAP-D3 overexpression results in a variable number of aggregates in salivary glands. GFP-dCAP-D3 aggregate numbers were examined in salivary glands (n= 61 cells) and the average presented. .
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Figure S4: dCAP-D3 depletion results in developmental delays and low body weights. A) Graph detailing eclosion rates based on daily total percentages of flies hatched from total number of wild type (w1118) and dCap-D3 mutant (dCap-D3c07081/c07081) larvae identified from 2 separate crosses. P-values were calculated using a chi-square test. p-value < 0.05. B) Bar graph showing average body weight in w1118 and dCap-D3c07081/c07081 males and females respectively. n = 5 replicates of 50 flies each, p<0.05.
Figure S5: Overexpression of dCAP-D3 does not affect gross nuclear organization during interphase.: A) DAPI staining of Drosophila S2 cells expressing GFP or GFP-dCAP-D3. B) GFP and GFP-dCAP-D3 salivary gland cells immunostained with an antibody against lamin C) Nuclear area measurements in GFP and GFP-dCAP-D3 salivary glands using lamin and DAPI. UAS-GFP: N= 5 glands, n=48 nuclei UAS-GFP-dCAP-D3: N= 3 glands, n =44 nuclei.
Figure S6: Overall correlation of RNA-seq samples. Principal component analysis showing that wing disc samples cluster more closely together and salivary gland samples cluster more closely together, based on gene expression signatures and dCAP-D3 expression status further partitions samples.
Table S1. RNA-sequencing data showing significantly deregulated target genes based on log2fold change > 1 (fold change > 2) and FDR < 0.05 in dCap-D3c07081/c07081 mutant wing discs and salivary glands, as compared to w1118.
Table S2. List of commonly deregulated genes between w1118 and dCap-D3c07081/c07081 mutant tissues
Video File S12. Live imaging performed in control (w; His2AvmRFP) wing disc. DNA is visualized in red.
Video S2File S3. Live imaging performed in dCap-D3 mutant (dCap-D3c07081/c07081; His2Av-mRFP) wing disc. DNA is visualized in red.
Video S34. Movie Depicting Prolonged Anaphase in dCap-D3c07081/c07081 mutant wing disc. DNA is visualized in red.
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