Fly Meeting 2012

Posted on March 7, 2012 by admin

Plenary Session I.

  • Thummel (Utah) – Regulation of energy metabolism in drosophila
  • Metabolic tissues and pathways conserved through flies. (e.g. insulin/glucagon balance)
  • Will develop obsiety and diabetes (including insulin resistance, cardiovascular disorders etc)
  • Transcriptional control by nuclear receptors: bind DNA (thyroid hormone receptors, steroid receptors, lipid metabolic receptors).
  • dEER mutants are lethal as larvae, dramatically reduced ATP as larvae. Glyocolysis enzyme production mostly off in these mutants.
  • Metabolic state of tumors, turn glucose into biomass and proliferation (RNA/DNA synthesis, protein/lipid synthesis). ‘Warburg effect’. Lots of nutrients available, need to increase mass. ‘aerobic glycolysis.
  • Effect of parental diet on metabolic health of F1 through F3 (i.e. epigentic inheritance). No mechanisms yet. (true for flies through mice and humans, true for maternal or paternal nutrient deprivation). DHR96 is parentally imprinted, required for maintenace of triglycerade levels (Rebecca Somer)
  • Flies don’t synthesize cholesterol.
  • Julia Simpson – neural fly grooming
  • Steve DiNardo (UPenn)
  • Niche stem-cells. Tstis stem cell niche. Hub cell (gametic stem cells). Hub cells specified late in embryogenesis from gametic precurosors
  • Notch required for early hub specification. Delta presented by mesodermal associated cells.
  • Role as stem-cells. Progeny are pushed away from niche, exit local JAK/STAT signaling, and differentiate. Hub cell is integrin anchored.
  • Is STAT activation sufficient for self-renewal stem cell behavior? – No, necessary but not sufficient.
  • STAT does drive self-renewal in the somatic lineage (cyst stem-cells). Cyst stem cells can drive germline stem-cells self renewal.

03/09/11

Regulation of Gene Expression

  • Barolo – patched-smoothened-hh signaling
  • ptc and pdd activated by hh signaling. Whever hh is activated ptc turns on. (localizing sequestration effect, negative feedback?)
  • dpp weak binding sites for Ci, need extra input to be bound? Strong Ci sites,
  • Replace weak with strong sites pattern change for dpp.
  • Improve affinity for wg find repression response instead of activation to high hh.
  • Many redundnat enhancers for ptc hh response.
  • Small lab (zhe Xu) patterning and timing of enhancers.
  • ID all bcd dependent enhancers in genome
  • Binding is better prediction for enhancer activity compared to cluster alone. Bcd binding site weakly informative (66 vs 38). New motif from bcd ‘positive’ enhancers. Looks like Zelda site.
  • Add zelda to negative enhancers, get (otd like) stripes.
  • Zelda sites facilitate bcd binding. Some bcd target enhancer not sensitive to zelda (otd). Kr (not bcd sensitive) is still Zelda sensitive.
  • Repressors limit bcd dependent activation.
  • Bcd strong activation of anterior genes, weak activation more posterior, more posterior genes inhibit anterior genes.
  • (see pending publication)
  • Furlong group.
  • Regulation of gene expression by CRM/ enhancers – link between organization and function
  • ‘homotypic synthetic CRMs’. Repeat a module several times. + pairwise combinations in different orientations and spacing. Tinman and with 4 partner TFs.
  • pMAD rpts synthetic CRM mimic dpp. Little bit wider in midgut.
  • Adding tinman in sense reomves viseral mesoderm pattern. Cadiac mesoderm from tinman input adds expression.
  • Not all the embryos have pattern and pattern not always complete. ‘penetrance’ Fraction of embryos that have any expression in the right region. ‘expressivity’ Fraction of signal that is in the correct domain.
  • Cardiac mesoderm more sensitive penetrance, expressivity more sensitive. Pmad tinman not sufficient for robst cardiac expression. Spacing, orientation and members effect
  • Tara
  • eve 3.7 4.6 fusions. Keep all stripe locations, modify levels as function of short-range repressor interactions. (can you actually identify the short range repressor connections).
  • Zelda (Rushlow lab)
  • important for early gene activation.
  • Zelda and AP / DV patterning. Antp much wider cc14 stripe in zelda null.
  • Zelda and rho fraction of nascent transcripts strongly dependent on Zelda.
  • Widen expression domain by adding Zelda sites. Do you add them in proximity to the endogenous dorsal sites.
  • Zelda corresponds with open chromatin states / highly occupied targets ‘HOT’ spots. Most enriched motif in HOT spots is CAGGTA (zelda). Local chromatin binding.

 

  • Cis-regulatory of hox-genes (Lohmann lab Heidelberg)
  • Dfd Chip-seq, ID dfd responsive enhancers. ID enriched motifs for spatially localized coregulators.
  • Output of Hox highly dynamic, based on spatially temporal cofactors.

 

  • Hox specificity (Richard Mann lab U Chicago)
  • Why have the precise expression if they all have the same binding properties. Don’t function only as monomers, binding properties changed by dimerization partners.
  • Exd and Hth bind with Hox genes.
  • Progressive gel shift, isolate bound fraction, deep sequence. Get range of affinities.
  • Scr and Ubx monomers bind same sequence.
  • Bind different sequences in dimers with Exd.

 

  • Rob Zinzen
  • chromatin signatures on enhancers before during and after activation
  • PolII K27me3.
  • K27ac, K79me3 and PolII only found on active enhancers,
  • separate intronic and intra-geneic enhancers? (chromatin mark of transcription vs. mark of enhancer per se).
  • Pol II links tightly to enhancer.
  • Snail as an activator (Leptin Lab)
  • Snail mutant, ectoderm in
  • snail as an activator or a repressor of repressors?
  • snail binds mesodermal enhancers. Snail and twist co-occupy enhancers.
  • (35 positive regulator: mef2 tin htl hbr, etc)
  • cell culture luciferase assay
  • Mef2.
  • Twi activates a repressor of snail.
  • Mutate snail binding sites. Twist stimulates mutant enhancer. Snail and twist stimulates expression.
  • 56 positive motifs.
  • Positive enhancer have dCTF motifs.

 

  • Yan bistable switch (Chicago)
  • Muscle and heart enhancer of eve locus.
  • Multiple nehancer required for yan contribution to eve repression for
  • Compare relative intensity of eve clusters upon deletion of multiple regulatory regions.
  • Deletion of one enhancer reduces binding of Yan at the other loci.

 

  • Campbell lab (Pitt)
  • Why recruit co-repressors
  • Brk uses CtBP and Gro.
  • CtBP represses some targets but not all, 3R repression domain needed for some not all.
  • Generate endogenous brk mutants. Make Brk attP knockout, and re-integrate brk gene at endogenous locus.
  • CiM and 3R mutants have 75 fertile but viable look lke wildtype. Double knockout is infertile.
  • Gro is required as sufficient
  • GRO is required for cuticle expression
  • CtBP – brk involved in egg shell patterning.
  • GRO downregulated my MAPKs during oogenesis? Requiring CtBP?

 

  • Albert Erives
  • Enhancer construction
  • Naked DNA, pol machinery can turn on gene independent of activator.
  • With chromatin, we see a threshold response of transcription to activator – suggests effect is de-repression, opening of chromatin. Transcription state is more or less binary.
  • Length of dorsal twist spacer is important for location of DV boundary (position of threshold).
  • One of the ways to change enhancer thresholds is to add redundant elements with different spacers.

 

  • Eve2 evolution. (Reinitz lab)
  • closely related species individual binding site predicted to be ancestral.
  • Further out species of sequences predicted to make a good pattern but not be an ancestor (and sites that are anscestral but no longer likely to make a good pattern).
  • Kr and Bcd have stronger inputs into sim eve2 than mel str 2, believed to be ancestral.
  • Trans changes to make vir str2 look better in mel. Suggest need just a bit more bcd.

 

  • Mathilde
  • More strongly bound peaks are more strongly conserved – weak binding is harder to detect, missed detection rate is higher.
  • Global expression comparison eml to pseudo – tightly conserved, more so than TF binding. Compenstory events balance out TF changes?

 Epigenetics and Chromatin

  • Ringrose.
  • Vg PRE and role of RNA in Pc
  • Vg PRE is transcribed in all tissues where vg is also expressed. Antisense, non-coding transcript is expressed before vg is expressed.
  • In brain the PRE mRNA is expressed in the vg OFF cells (Pc repressed). Forward strand transcribed
  • Make transgene vestigal PRE.
  • Ectopic transcription of trangene PRE activates endogenous locus. Add PRE see fewer 3D FISH loops than transgene insert without a PRE.
  • Transcription of PRE under GAL4 represses the endogenous locus
  • RNA memory mechanism
  • transinduction due to paring creates supressor vof variagation?
  • Or RNA diffusable factor?
  • Pirrota
  • Insulators bring active genes into transcription factories and repressed genes to PcG bodies.
  • McP and PRE have an insulator part and PRE part. Tag transgene with 128 copy lambda repeat, cross to GFP LacI. Look for one dot cells. Delete insulator part, see less 1 dot.
  • Combine with RNA FISH measure transcriptional state
  • Transcription contributes to co-localization of transcripts in the eye. PRE part is
  • Trx decreases colocalization in expressed tissue.
  • Silenced transgene colocalize with PcG body. Active colocalize to Pol-II foci.
  • Ant-AbdA interaction depends on CTCF (based on CTCF knockdown).
  • PcG bodies decrease size under CTCF knockdown
  • Genetic screen for Polycomb group mutants
  • Pairing sensitive silencing (only in homozygotes is region silenced.
  • 4 pairing silenced copies of w+. Use FLP to create mutations just in the eye (not necessary for viability /fertility). 32 mutations in known PcG genes which lead to surpession of pairing.
  • 18 new genes. 2 code for tRNA ammino sythetases. Charge tRNAs.
  • mutant is a hypomorph. Recessive. Single amino acid change.
  • RNAi mutants do not affect pairing sensitive silencing.
  • Welcome Bender (Harvard)
  • Mapping chromosal proteins of bithorax complex in single parasegments
  • 8th AB segment requires all of the BX-C. T2 requires none.
  • Mod-encode H3K27me3 all segments together – whole region. S2 pattern doesn’t make sense.
  • Gal4 Gal80 overlap to get GFP in subset of cells.
  • Also use Hox enhancers with engrailed promoter + PRE to maintain expression after gaps are lost. Make lines which express GAL4 in each parasegment: 4 5 6 and 7.
  • FACS sort, get 10^5 nuclei. For CHIP. K27me3 still throughout whole region in PS5 and PS7.
  • Nurse cells undergo endoreplication and then swtich from partially condensed “Polyblock” state to a decondensed state (called polyblock dispersal). Otu mutants fail to leave polyblock state.
  • Peanuts gene loss of function enhances the otu phenotype. Splicesome component. Required for splicesome recycling. Loss of recycling results in progressive loss of gene expression.
  • HP1 evolution. Mia Levine
  • 5 Known HP1 genes. Get 5 new ones plus large number with just the chromo domain or just chromo-shadow domain. Most new genes show biased testes expression. Gene replacement in HP1 across many species. HP1E essential for male fertility. Sterility manifests after sperm entry into seminal vesicle. Paternal effect gene.
  • Elgin Active genes on Chr4
  • heterochromatic. High repeat density. H3K9me2/3 and Hp1 colocalization.
  • 80 genes in distal 1.2 Mb that are transcriptionally active. H3K4me2 marks promoters.
  • H3K9me3 becomes silencing mark. H3K36me3 becomes active associated instead of silent.
  • H3K9me2 not at promoters. H3K36me3 promotes histone deacetylation. HP1a promotes demethylation of K36. Propose dynamic Hp1, K36me3….
  • Deplete Hp1a = less expression. Genes start to pause at promoter.
  • Loss of POF (viable): HP1 remains associated on heterochromatin of 4, but is lost on the expression region. New mechanism of localizing HP1 to 4th chromo. Most chromo4 p element insertions → variagation. Any insertion near repititious transposable element 1360.
  • Demonstrate 1360 generates heterochromatin using insertion with reporter.
  • 1360 has piRNA hotspots necessary for this heterochromatic silencing.

Systems Bio Section

  • Nicolas . Carthew and Amaral lab. Yan Network.
  • Yan – bistable system: Yan under Notch activation = pluripotent state.
  • MiR-7 + Pnt-P1 and MAE all repress Yan in the other state.
  • How is robust differentiation maintained despite variation in network components.
  • Yan-YFP 35 kb BAC: rescues null transheterozygote.
  • Colabel nuclei with His-RFP to compare intensities.
  • Dynamics of Han, induction, high sate, slow decay. (Protein dynamics).
  • During induction increase in variation of Yan.
  • During slow decay phase variation reduces.
  • Eye patterning normal 2x through 6x yan.
  • Not a stable limit to Yan concentration. 6X is much brighter. (yan self repression?)
  • readout of Yan is saturated. Do you know the cis regulatory targets of Yan?
  • If Yan network is bistable, there is presumably some level x

 

  • Rupinder (Arnosti Lab)
  • Quantiative models of enhancer function.
  • 300 bp rho NEE. Dorsal twist and snail. 1.7 kb upstream. Systematic perturbation knock out single activators, double activators, or repressors. Co stain snail and eve.
  • Any dorsal site knockout drops expression by 40%.
  • Different double knockouts have different strnghts of knockout.
  • Knockout snail 1 and snail 4, no obvious effect. Overlapping twi site doesn’t matter.
  • Stat mech state model. Survey different cooperativity models (all sites, near sites etc).
  • Compare all models on data.
  • Sog, without Zelda sites, does your model predict observed reduction

 

  • Alexi Brooks (Raftery Lab) BMP gradient
  • Using pMAD staining as readout of dpp activity gradient.
  • Natural variation in size of Drosophila wing disk. pMAD gradient scales with disk size.
  • Use ras to perturb wing pouch size.
  • Lambda (decay rate) varies substantially with DV position. Sample size not easily large enough to detect a 10% difference.

 

  • Yorgi Jaeger
  • Gene network interactions shape patterns (Gap genes)
  • Sum weighted contribution of all reglatory inputs, then threshold (sigomoid) that. + diffusion and decay. Do genetic selection on model parameters to fit the data.
  • Reproduce anterior shift. Model predicts that hb posterior stripe activation + cross repression drives anterior shift. Cross repression also predicted my model to be source of sharpening.
  • Megaselia abdita – no caudal gradient, still has bcd. Has D mel like gap pattern.
  • Clogmia albipunctata (moth midge). No bcd (something else) + cad, reduced gap network, 6 eve stripes.
  • Hard to do protein stains in other species, mRNA works better. Can still use mRNA traces to recover de novo the same gap gene network / interactions.
  • Argues that post-transcriptional regulation not substanatially important.

 

  • Manu
  • Eve-YFP and Eve2
  • Flanking binding sites to minimal eve2 not required for viability (despite evolutionary conservation).
  • Border position dynamics are changed in min eve2.
  • Non-genetic variation in the order of boundary formation (which stripes peak first). Sometimes 2 peaks 2nd, sometimes 3 peaks second. MSE much prefers 2 first (lacks some repressors)
  • Early eve is sex specific, Male has lower repression between stripes 1 and 2. Final segmentation is not.
  • Dosage response with gt. Gt-YFP. 1 dose gt also shows loss of repression – gt is not fully dose compensated.
  • Consequences of sex specific eve expression. Lose sex specific eve expression in MSE line – MSE rescue males are less happy.

 

  • Miriam (Stas lab)
  • Dorsal appendages in drosophila eggshell. Floor cell edges constrict, sliding over neighbors, to form a rosette. Smooth, straight boundaries to facilitate these movements ncessary. Caused by tension in tissue. MyosinII upregulated on apical surface. Cables of Myosin along boundary.
  • Baz is perpendicularly organized relative to MyosinII.
  • Vertex model of dorsal appendage formation explain cell shapes seen and formation of buckle and the twist of the floor cells beneath the roof cells.

 

  • NADPH producing enzymes
  • some enzyme activities compensate reductions in other enzyme activities, some don’t.
  • Changes as evidence of successful adaptation to stress as opposed to signs of the system breaking down.

Sunday Plenary

  • Oogenesis (live migration of pole cells). Can steer cells with photo-activatable Rac, induces phillapodia formation. Can make them turn around.
  • Reversing apoptosis: treat cells with ethanol – phenotypically shrivel, form ‘blebs’, recover after washing.
  • Elecrophoresis cells in an agarose gel. Untreated cells don’t have breaks, DNA stays together.
  • Apoptoic EtOH treated cells have double strand breaks and generate ‘commet tails’ of DNA.
  • Treated and washed cells keep DNA together, have repaired double stand breaks.
  • Some repairs lead to DNA damage (incorrect DS break repair).
  • Anastasis ‘rising to life’
  • Tether Gal4 to membrane with a caspase linker. UAS-FLP will activate constitutive GFP, mark the ‘recovered’ cells.

 

  • Manyuan Long (Evolution of New Genes)
  • New genes by gene duplication. Duplication and hoping into existing genes.
  • 220 new genes since Dw. (36 m yr)
  • 2/3rds gene duplication. Much gene duplication by reverse transcription” intronless, poly-A intact, has short flanking duplicates. X –>A retroposition bias.
  • Many new (3-6 m.y. old) genes expressed in brain. Also true for new human genes
  • Sibling pardox (how do the species w/o the new gene survive?) Network rewires much more rapidly.

 

  • Julie Brill PIPs control cell morphogenesis in Drosophila
  • Function of PI4 kinases PI4KaIII essential
  • Fwd not essential. Over-express PIP phosophatase in male germ cells. Lose PIP membrane localization. Required for cytokensis. Required for spermatid elongation. Lose nuclear polarity localization and microtubule organization.
  • Low sigD (phosophotase) still do cytokensis and elongation, but mislocalize the nuclei and are sterile.
  • Axoneme organiztion loses polarity.
  • PiP2 mutant doesn’t have as dense chromatin around outside of nuclei.
  • PIPs link nucleus to microtubules
  • To test: just describe the shape changes of nuclei that correspond to gene activation from early to later development.
  • Canoe shaped nuclei lose all histones in male testes (geometry of late nuclei) .

 

  • Furlong
  • CRM identification mostly solved. How do we understand these.
  • DNA sequence to TF occupancy (largely unsolved)
  • 80% of predictions from chip binding give activity in the predicted cells / predicted times. e.g. Twist for early mesoderm, not twist in late mesoderm.
  • One pattern can be generated in many ways. Challenge to generalizable grammar.
  • Cardio enhancers
  • Only cardio cells express Tin, Doc & Pnr. ID 4,000 cardio enhancers from ChIP for these factors.
  • Mostly find all 5 TFs on most enhancers. More so than observed for other groups of TF chips.
  • Is this collective occupancy mesoderm specific? When tinman is lacking, other 4 or lacking.
  • ‘no evidence for consistant motif grammar’
  • orientation, spacing, number of motifs no pattern for cardiac enhancers.
  • Factors recruited to multiply occupied CRMs without need of motif. Once most of TFs are there the extra ones bind just fine.
  • The ‘TF Collective’ model instead of the ‘Enhancesome’. Doesn’t require motif to be present for all factors.
  • ‘Provocative conclusion’: no enhancer grammar.
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