Sarah Elgin, Anya Goodman, Wilson Leung

Finding Genes in a New Fly Genome: Teaching about Genes/Genomes via Bioinformatics Research
Sarah Elgin, Anya Goodman, Wilson Leung Eric Tsoi, Charlene Emerson, David Carranza January 2012 Graphics: Karmella Haynes

Workshop Goals Introduce Genomics Education Partnership
Hands-on practice with genome annotation Discussion of curriculum options 3-week module ( ~10 hr: 1 lecture, 3X3 lab) 5-week – add a more difficult project 10-week - real research! Scientific background on Drosophila genome Next workshops: June 24-26, August 19-22 (HHMI supported) Source: SCR Elgin

Genomics Education Partnership (GEP)
Source: GEP website, Slide: Wilson Leung Notes: GEP members through 2011. Goal: to engage students in a genomics research project, via a research-oriented AY lab course. Work organized through the GEP website.

Our GEP research goal: Use comparative genomics to learn more
about heterochromatic domains, analyzing the dot chromosomes and a control euchromatic region of Drosophila genomes Status Reference Completed Source: Evolutionary tree from FlyBase. Slide: Wilson Leung Notes: The research accomplished by GEP students. What can we learn from the comparative genomics of the dot chromosome? Initially we have looked at gene and chromosome evolution in several distant species (ca. 40 mil yr); this year we are also starting work on D. ananassae, where the dot chromosome has undergone an unusual expansion (10-fold larger – all repeats!). Annotation Sequence Improvement New Project FlyBase:

I hear and I forget. I see and I remember
I hear and I forget. I see and I remember. I do and I understand Confucius The scientific method allows ordinary people to do extraordinary things. Francis Bacon Genomics provides terrific opportunities to engage undergraduates in research! Slide: SCR Elgin Source of picture of Confucius –internet. The saying may be older than Confucius, but Google gives him credit.

Strategy: divide and conquer! The D. mojavensis dot chromosome
Students completed 68 projects covering 1.7 Mb closing 26/28 gaps, adding ~15,000 bp and improving ~5000 bp. Each project finished and annotated (all isoforms) twice; reconciliation for quality control done at Wash U Fosmid sequence matches consensus sequence Slide: SCR Elgin Source: project organization for the GEP by C Shaffer and W Leung. Notes: Colored blocks indicate the overlapping set of fosmids used to improve the sequence of the D. mojavensis dot chormosome. Putative polymorphisms Finished sequences submitted to Genbank, annotations to Flybase.

Genomics Education Partnership
4/25/2017 Annotation: Create gene models using sequence homology and computational predictions Finished Sequence Sequence Homology Gene Predictions Slide: W Leung Source: Mirror UCSC browser site used by the GEP. New! RNA seq tracks! Copyright © 2008, Washington University in St Louis

Learning from the annotation process…
Student Gene Predictors Become familiar with tools available for finding genes; manage data Identify genes, create best models (start, exons, stop etc.) Requires synthesis of multiple lines of evidence Use power of comparative genomics; reference D. melanogaster Analyze genome organization (synteny), repeats Address questions of evolution Experience presenting data, supporting conclusions based on available evidence Students are making an original contribution Each project done twice independently, 60% - 75% congruence Source: Chris Shaffer Slide: SCR Elgin Notes: The four gene predictors used on our browser predicted four different exon assemblies for the gene under study. Note that all four gene predictors missed the first two exons, which are rather short. The GEP student was able to identify these, working from the D melanogaster sequence and searching for an upstream region with this coding capcity.

Genomics Education Partnership, Alumni Workshop
June 2008 But are they learning? GEP annotation quiz results, Mean Annotation Score Source: D Lopatto, Grinnell College Pre-course A Pre-course B Post-course A Post- course B We see a positive correlation between quiz scores and self-reported gains. Copyright © 2008, Washington University in St Louis

June 2008 GEP assessment: CURE survey (D Lopatto) Tackling real problems Understanding the research process Lab techniques Source: Lopatto et al, 2008, Science 322: For most gains, a GEP semester course is as effective as a summer research experience! Copyright © 2008, Washington University in St Louis

Selected quotes….. The class was very intellectually challenging for me. It taught me to think in a way that I had never thought before. The thing that sparked me the most was the fact that I was able to perform the BLAST searches on my own and was able to explain to my instructor what I had done. I seriously think this should be the model for all biology courses. I guess we learned about genomics through doing the tasks…. it was sort of a self-teaching class…. Everything we gained from the class…was either found by desperately messing around on the various websites …or by talking with other students. I know if I could survive this class then I could survive just about anything. Source: SCR Elgin, D Lopatto Notes: 80% of the comments on anonymous surveys were positive, 20% were not. Initial frustration was dissipated when the students were convinced by their own success.

Some things to look for while annotating your dot chromosome genes….
Is there a homologous gene in D. melanogaster? Is it on the dot chromosome? Are all of the isoforms found in D melanogaster present? How many exons? Any unusual splice sites? Can you identify the TSS? What is the order and orientation of genes compared to D. melanogaster? Are there repetitious elements nearby? Check out your gene on FlyBase – what is the pattern of expression in D. melanogaster? Has a function been described? Pooling our data, can we see any common 5’ or 3’ motifs unique to dot chromosome genes? Slide: SCR Elgin.

The Genomics Education Partnership: Investigating the and Function of the Dot Chromosome Genes in Drosophila Sarah C R Elgin January 2012 Graphics: Karmella Haynes.

Goal: to understand the organization and functioning of the dot
A collaborative investigation involving: - former members of the Elgin Lab: Lee Silver, Carl Wu, TC James, Joel Eissenberg, Lori Wallrath, Fang Lin Sun, Karmella Haynes - current members of the Elgin Lab: Nicole Riddle, Tingting Gu, Chris Shaffer, Wilson Leung - modENCODE: Gary Karpen, Mitzi Kuroda, Vincenzo Pirrotta, Peter Park, and their colleagues - Faculty and students of the Genomics Education Partnership Goal: to understand the organization and functioning of the dot chromosome in Drosophila, an unusual heterochromatic domain. Funding: HHMI Professors Program NIH General Medical Sciences, National Human Genome Research Institute

Minimum haploid DNA content - the C Value Paradox
Genomics Education Partnership 4/25/2017 Minimum haploid DNA content - the C Value Paradox Instructor: Professor Sarah CR Elgin Slide: Gabriella Farkas Sources: Gene Regulation for Higher Cells: A Theory. R Britten & E Davidson, Science, 1969 Jul 25;165(891): Notes: The genomes of eukaryotes have a surprisingly large amount of DNA. It has been suggested that this is related to the “complexity” of the organism- but complexity is hard to define! (Note the lack of a Y axis in this representation, published in Science!) Britten and Davidson, 1969 Science 165:349 Copyright © 2011, Washington University in St. Louis

4/25/2017 Larger genomes reflect high levels of repeats - retroviral and DNA transposon remnants (TEs) This introduces many complications when assembling a genome sequence! Source: CD Allis, T Jenuwein, D Reinberg, Overview and Concepts, in “Epigenetics” (2007)ed Allis, Jenuwein, Reinberg, Caparros, Cold Spring Harbor Laboratory Press, NY. Notes: Simple eukaryotes such as yeast often have a small genome in which the protein-coding genes are the major fraction. However, genomes of higher organisms are primarily made up of repetitious DNA and non-coding sequences, probably derived from repeats. Thus the increase in genome size appears at least in part to be the consequence of retroviral and transposon invasion of the genome, as well as local duplications etc. Allis et al: Epigenetics 2007 Copyright © 2011, Washington University in St. Louis

Conserved noncoding - regulatory? 3.5%
Eukaryotic genomes are very large – and most of that DNA is non-coding! Human Genome 3 Gb ~2 m/cell ! Coding exons 1.5% Conserved noncoding - regulatory? 3.5% TEs – retroviruses, DNA transposons Slide: Xiaohui Xie, MIT (2007). Notes: Is all of this repetitious DNA garbage – would we eliminate it from our genome if we could? Or is it junk? Something that might prove useful in the future? Repetitious sequences are used in chromosome structures such as the centromere, and there is evidence that at least some regulatory sequences (perhaps many) derived from repetitious sequences. But we must insure that transposable elements remain quiescent – not transposing, which would result in mutations. The DNA must be packaged to fit into the nucleus, and that same packaging into chromatin may be the key to silencing. Key Questions: Is it junk or garbage? How is DNA packaged into a nucleus? How is silencing maintained – while allowing appropriate transcription ?

Chromatin formation: First step - packaging in a nucleosome array
Second - differential packaging into heterochromatin & euchromatin DNA Chromatin Histone protein core Source: Left, Lodish et al, Molecular Cell Biology, 4th edition; right, Felsenfeld et al, (2003) Nature 421:448. Notes: Left, a generic illustration of a eukaryotic cell. Note the clumps of denser material in the nucleus, referred to as heterochromatin; these contrast with the less dense nuclear material, the euchromatin. Right: a condensed metaphase chromosome being gradually unraveled to reveal a chromatin fiber. DNA does not exist as a free molecule in the nucleus. It is packaged into chromatin which is defined as DNA plus its associated proteins. The basic subunit of chromatin is the nucleosome, which is 146 bp of DNA wrapped around a histone octamer core. Note that while we have good data on nucleosome structure, the illustrations of higher order packaging, while consistent with current data, are not well established. Chromosome (metaphase) Lodish et.al., Molecular Cell Biology, 4th Edition Felsenfeld et al. Nature 2003, 421: 448

Electron micrograph of chromatin fibers (rat thymus nucleus)
Genomics Education Partnership Electron micrograph of chromatin fibers (rat thymus nucleus) 4/25/2017 Instructor: Professor Sarah CR Elgin Slide: Gabriella Farkas Sources: based on Figure 1. from Olins AL,Carlson RD, Olins DE.. J Cell Biol Mar;64(3): Visualization of chromatin substructure: upsilon bodies. Notes: Prior to 1970, most biologists thought that the DNA in the eukaryotic nucleus might exist in two forms- relatively accessible (naked), if transcriptionally active, but inaccessible and coated with histones if inactive. This model of a chromatin fiber with DNA on the inside, histones on the outside, was turned inside-out by a series of experiments carried out in the early 1970’s. Some of the first evidence leading to the new model was electron micrographs such as that shown above, showing chromatin fibers released from a nucleus, with the appearance of beads on a string. We now know that the DNA is initially packaged by association with the core histones to form nucleosomes, with the DNA wrapped around the outside of the histone “bead.” This creates a fiber of ca. 10 nm diameter, referred to as a “string of beads,” or more properly, “string wrapped around beads.” 0.1 mm Olins et. al., 1975 J. Cell Biol, 64:528 Copyright © 2011, Washington University in St. Louis

4/25/2017 “A eukaryotic chromosome made out of self-assembling 70A units, which could perhaps be made to crystallize, would necessitate rewriting our basic textbooks on cytology and genetics! I have never read such a naïve paper purporting to be of such fundamental significance. Definitely it should not be published anywhere!” Instructor: Professor Sarah CR Elgin Sources: K D van Holde, “Chromatin”, 1989, ISBN , Springer-Verlag, New York. Notes: Such pictures were obtained both by Olins & Olins (Oak Ridge Natl. Lab.) and by Chris Woodcock (U. Mass. Amherst). However, publication was slowed by reviews such as that above. The reviewer correctly recognized that the pictures suggest a paradigm shift in our thinking about the chromatin fiber, and is concerned that the electron micrographs may be showing an artifact. However, on-going experiments by others showed that the subunits (nucleosomes) could be isolated and characterized, and indeed these subunits have been crystallized – although it took many more years effort to do so. Anonymous review of paper submitted by C.F.L. Woodcock, 1973, showing EM pictures of nucleosome arrays. Quoted in “Chromatin” by K.D. van Holde, 1989 Copyright © 2011, Washington University in St. Louis

The structure of the nucleosome core
Genomics Education Partnership 4/25/2017 The structure of the nucleosome core Resolution: 2.8 Å Half of the nucleosome structure is shown One turn of the DNA helix is visible (73 bp) View is down the superhelix axis Protein - DNA contact: white hooks Instructor: Professor Sarah CR Elgin Slide: Gabriella Farkas Sources: Figure 1 from: Chromatin structure: The nucleosome core all wrapped up. Rhodes, D., Nature 389, (18 September 1997); after Luger et al, 1997, Nature: 389: (18 Sept 1997) Notes: The illustration shows how DNA is bound and organized by the histone core. After 7 yrs effort, Tim Richmond and his colleagues crystallized the intact nucleosome core with DNA, giving a high resolution structure (2.8 A). To get good crystals he had to use an inverted repeat DNA (symmetrical around the dyad axis) and histones prepared in E. coli, to avoid any post-translational modifications (see below). Note the numerous DNA-histone contacts (white hooks) – this is a very stable structure, maintaining dsDNA, blocking transcription. Rhodes, 1997 Nature 389:231, after Luger et. al., 1997 Nature 389:251 Copyright © 2011, Washington University in St. Louis

DNA packaging domains Euchromatin Heterochromatin Less condensed
Chromosome arms Unique sequences; gene rich Replicated throughout S Recombination during meiosis Heterochromatin Highly condensed Centromeres and telomeres Repetitious sequences; gene poor Replicated in late S No meiotic recombination Slide: Gabriella Farkas. Notes: While all DNA is packaged into nucleosome arrays, the nucleosomes vary in the post-translational modifications of the histones and in the associated proteins. So while the distinction between euchromatin and heterochromatin was first recognized by the cytology (less dense vs. more dense regions in the nucleus when staining the DNA), and expanded to the collection of properties given on the slide, we are now able to define heterochromatin in biochemical terms. Specific residues in the histones’ amino-terminal tails are enzymatically modified, most often by acetylation, methylation, and phosphorylation. These modifications influence chromatin packaging and the level of gene activity at different regions of the genome. Transcriptional activators Hyper-acetylated histone tail Heterochromatin Protein 1 complex Hypo-acetylated histone tail; methylated H3/K9

Chromatin structure = epigenetics !
What sets and maintains tissue-specific gene expression patterns? Differences are heritable through mitosis, but independent of DNA sequence. DNA modification (mC) Chromatin structure Nuclear localization It’s all about silencing! How is chromatin assembled? When, where and how does gene silencing occur? Incorrect silencing can lead to genetic disability, as seen in Fragile X syndrome Zoghbi and Beaudet 2007 Slide: Illustrations from Nicole Riddle Source: Fragile X Foundation; Zoghbi and Beaudet, 2007. Notes: The basic question of interest is how is chromatin assembled and modified to maintain appropriate levels of gene expression. In particular, heterochromatin formation biases to gene silencing. And of course it must be done correctly. FRX is caused by a trinucleotide repeat expansion in the 5’ regulatory region of FMR1, that then causes hypermethylation and silencing of the gene. Kids with FRX have big ears and high foreheads and elongated facial features, and have some level of mental disability. Fragile X Foundation 23

1 2 Heterochromatin formation – silencing counts!
How is heterochromatin organized and packaged to promote silencing? 1 2 The fourth chromosome appears heterochromatic but has ~80 genes: - do these genes have unusual characteristics? - how has the chromosome evolved? -- how do these genes function?

Fruit Flies! Short life cycle, easily maintained: good genetic tools
Polytene chromosomes: excellent cytology Biochemical approaches Simple genome, good reference sequence PEV – reporter for gene silencing, heterochromatin formation Metazoan useful for behavioral, developmental and human disease research Mary Lou Pardue, MIT Slide: Polytene chromosome showing in situ hybridization from Mary Lou Pardue, MIT. Notes: The fruit fly is a great model organism for epigenetic studies: It’s short life span and simple genome (4 chromosomes) facilitates genetic studies; phenotypes easy to score; biochemical approaches are possible; the salivary gland polytene chromosomes provide a pretty high resolution visualization of the chromosomes, allowing studies of the distribution of DNA sequences (in situ hybridization) and proteins (immunofluorescent staining). We can use a reporter gene (white) to monitor the chromatin environment; when this gene is in its normal euchromatic environment, it is fully expressed giving a red eye, but when moved to heterochromatin (by rearrangement or transposition) it is silenced in some of the cells in which it should be active, resulting in variegation. Many of the deleterious mutations that cause health problems in humans can be modeled in the fruit fly, including Fragile X. (Flies are relevant!) euchromatin heterochromatin expressed silenced

Transposition of a P element reporter allows sampling of euchromatic and heterochromatin domains
X 2L 3L 2R 3R 4 Silenced 1% Source: L Wallrath & SCR Elgin (1995) Genes Dev 9: 1263 – 1277. Notes: The P element construct used is diagrammed above. The hsp70-driven white gene gives us the eye phenotypic marker; the hsp26 gene marked with plant DNA has been used for the chromatin structure studies that follow. In situ hybridization showed that the variegating P element inserts all map to the pericentric heterochromatin, telomeres, Y, or fourth chromosome, all previously known heterochromatic domains (not to scale). Active 99% And the Y chromosome Wallrath and Elgin, 1995

Assessing chromatin structure- same gene, different environments Analysis based on nuclease digestion of chromatin Slide: Gabriella Farkas Source: Based on experiments in FL Sun, M Cuaycong, SCR Elgin (2001) Mol Cell Biol 21: 2867 – 2879, and D Cryderman, H Tang, C Bell, DS Gilmour, LL Wallrath (1999) Nucleic Acids Res 27: Notes: Now we can ask what happens to a gene that is normally in euchromatin when it is placed in a heterochromatic environment. It is being silenced inappropriately – is this due to a change in chromatin packaging? We can carry out chromatin mapping studies using nucleases to examine the chromatin structure of the native gene. Digesting with micrococcal nuclease will reveal the nucleosome pattern, while digesting with DNase I will show where DNase hypersensitive sites – nucleosome free regions – are present. We find that when a gene is moved from a euchromatic to a heterochromatic domain, the nucleosome array becomes much more regular, with the loss of DH sites. The absence of DH sites most likely contributes to the observed silencing. The euchromatic hsp26 transgene: - DH sites: accessibility at the TSS, upstream regulatory region irregular nucleosome array The heterochromatic hsp26 transgene: - loss of DH sites - regular nucleosome array

Looking for heterochromatic proteins by immunofluorescent staining of the polytene chromosomes: discovery of HP1a James & Elgin,1986; James et al 1989 Source: TC James & SCR Elgin (1986) Mol Cell Biol 6: 3862 – 3872; and TC James et al (1989) Eur J Cell Biol 50: 170 – 180. Notes: To look for proteins preferentially associated with heterochromatin, we prepared monoclonal antibodies against proteins that bind tightly in the nucleus, and screened by using the antibodies for immunofluorescent staining of the polytene chromosomes. Heterochromatin Protein I (HP1) is preferentially associated with the chromocenter, small fourth chromosome, telomeres, and a few sites in the chromosome arms. (Note that in polytenization, the pericentric heterochromatin is under-replicated, and all of the euchromatic chromosome arms fuse in a common heterochromatic chromocenter, C above.) C C HP1 Phase

Heterochromatin-associated gene silencing is dependent on HP1
Mutations in Source: Eissenberg et al (1990) Proc Natl Acad Sci USA 87: 9923 – 9927. Notes: This experiment provides key genetic evidence that HP1 plays a role in the mechanisms of silencing; loss of HP1 (either a truncation mutant or a mutation in the chromo domain) has this dominant phenotype, a loss of silencing in a reporter showing a variegating phenotype. The original mutation was recovered by T Grigliatti in a screen for mutations with this phenotype – suppression of variegation. Eissenberg et al then sequenced the DNA from these flies, proving that the mutation is in a gene coding for HP1a. The homozygous mutation is lethal, showing that HP1 is an essential protein. gene for HP1a Mutations recovered by T Grigliatti as suppressors of PEV. Dosage dependent response. Eissenberg et al, 1990, PNAS 87: 9923

HP1 interacts with both the modified histone H3K9me2/3 and the modifying enzyme
Chromo Shadow SU(VAR)3-9 HMT Sources: Tschiersch et al (1994) EMBO J 13: 3822 – 3831; Lachner et al (2001) Nature 410: 116 – 120; Bannister et al (2001) Nature 410: 120 – 124. Notes: How might heterochromatin be assembled? Work in mammalian cells showed that the chromo domain binds H3 iff it is methylated at K9 (Jenuwein, Kouzarides). The shadow domain generates HP1 dimers, forming a platform that binds several other chromosomal proteins. Among these is SUV3-9; Jenuwein showed that the human homolg has an enzymatic activity that methylates histone tails at lysine 9. Thus HP1 can both recognize a histone modification (H3K9 methylation), and promote that modification by interacting with the enzyme. This suggests a mechanism of recruit/methyl/recruit that could be used both in maintenance and propagation of heterochromatin. Note that because of its dimerization through the CSD, HP1 could work as a bifunctional crosslinker. Histone 3 methyl-Lys9 H3 K9 methylation [(SU(VAR)3-9 identified in screen by Reuter; H3 interaction first shown from work in mammals – Jenuwein, Kouzarides; demonstrated in flies by Imhof.]

Model for spreading of heterochromatin
Slide: Gabriella Farkas, SCR Elgin lab. Notes: Animation of spreading of heterochromatin: HP1 recognizes H3K9me3 and binds; it then recruits SU(VAR)3-9, which can modify the next nucleosome accordingly.

Establishing silencing: a multi-step process
Loss of euchromatin marks Gain of heterochromatin marks Source: Elgin, SCR & Reuter, G (2007) in “Epigenetics,” ed Allis, Jenuwein, Reinberg, Caparros; Cold Spring Harbor Laboratory Press, New York. Notes: Of course it is not that simple! Over 100 Su(var) mutations have been reported – over 100 proteins needed to generate / maintain silent chromatin! Some of these are needed to remove the “active marks” – for example, dLSD1, the enzyme that demethylates H3K4. And some are required to add the “silencing marks”, as well as being a structural component of the heterochromatin. Thus a cascade of events is required to shift a locus from a euchromatic, activatable status to a heterochromatic, silenced status. Remember – these processes are reversible – but seem to be fairly stable changes during differentiation, as a specific cell type acquires a limited set of active genes. wm4 reporter (screens by Reuter, Grigliatti, others)

2 Heterochromatin formation on the dot chromosome…
The fourth chromosome appears heterochromatic but has ~80 genes: - do these genes have unusual characteristics? - how has the chromosome evolved? - how do these genes function? 2

The Drosophila melanogaster fourth chromosome exhibits an amalgam of euchromatic and heterochromatic properties (HP1a association) Heterochromatic properties: late replication, lack of recombination high repeat density (30%) antibody staining of HP1, H3K9me2/3 But… the fourth has ~ 80 genes in distal 1.2 Mb these genes are transcriptionally active! James & Elgin,1986; James et al 1989 Slide: Wilson Leung Source: James & Elgin (1986); James et al (1989); see previous slide. Notes: The fourth chromsome of D melanogaster (sometimes called the “dot” chromosome from its metaphase appearance, but more properly known as the Muller F element) appears entirely heterochromatic by classical criteria, but the distal 1.2 Mb has 80 genes, a gene density similar to that found in the euchromatic arms. C C HP1 Phase

Most hsp70-white reporters exhibit variegation
on insertion into the fourth chromosome 2-M1021 39C-12 2-M390 39C-52 Source: Sun et al (2004) Mol Cell Biol 24: 8210 – 8220, and Riddle et al (2007) Genetics 178:1177 – 1191. Notes: A map of the fourth chromosome distal portion (centromere to left) showing the presence of TEs and the genes (bars below). Each triangle indicates a line carrying a single P element reporter inserted at that site, with the eye phenotype shown, red or variegating, indicating a euchromatin or heterochromatin environment. Most fourth chromosome genes lie in a domain that appears to be heterochromatic, at least in the eye primordia. Note that the heat shock promoter used here is active/activatable in almost all tissues. Sun et al 2004; Riddle et al 2007

The Drosophila dot chromosomes are typically 25% - 30% repetitious DNA
( – but up to 80% in D. ananassae! ) Source: Leung et al (2010) Genetics 185: 1519 – 1534. Slide: Wilson Leung Notes: Our first published analysis is a comparison with D. virilis. We find that both D melanogaster and D virilis dots have similar levels of repetitious DNA (27-30%), intermediate between that found in the euchromatic arms (~10%) and that found in the pericentric heterochromatin (~60-70%). Note that our ability to find repeats depends on having good libraries of repeat sequences for a given species. The commonly available libraries are based on D melanogaster; since other species have been invaded by different retroviruses and transposons, their repeats may be underrepresented unless a special effort is made to find these repeats. Looking at the types of repeats, we see a higher relative concentration of DNA transposons (red) on the dot chromosomes and a higher concentration of retroviral sequences (green) in the heterochromatic regions. However, many repeats remain unclassified (blue). GEP, 2011

Dot chromosome genes: introns are larger, exons show less codon bias
Euchromatic Leung et al Genetics 185: Codon Bias Dot Heterochromatic Source: Leung et al (2010) Genetics 185: 1519 – 1534. Slide: Wilson Leung Notes: We find that the dot chromosome genes on average are larger than genes in the euchromatic arms (8 kb vs 2 kb) and have more introns (8 vs 3). Most of the size difference can be explained by more and larger intron sizes due primarily to repetitious DNA. (The graph shows the percentage of introns that are a given size (x axis) or smaller.) We also find that codon bias is reduced on the dot chromosomes, presumably because the lack of crossing over. (Codon bias refers to the use of codons – in theory an organism could use all 64 codons, but in practice most organisms use fewer.) Intron Size D. melanogaster Dot D. melanogaster Euch. D. melanogaster Het. D. virilis Dot D. virilis Euch.

Initial analysis of Drosophila virilis dot chromosome fosmids
Genomics Education Partnership 4/25/2017 Initial analysis of Drosophila virilis dot chromosome fosmids Sources: From Slawson et. al., Genome Biol. 2006; 7(2): R15. Notes: While most of the same genes are found on the dot chromosomes in D melanogaster and D virilis, there have been extensive rearrangements, presumably by inversions. For example, pan and Caps are on opposite ends of the dot in D melanogaster, but are found on the same fosmid (#30, dark blue) in D. virilis. In contrast, the gene cluster of fosmid #103 (turquoise) has maintained the same order and orientation – perfect synteny. Almost all of the same genes are present (~90%), but rearrangements within the chromosome are common – a minimum of 33 inversions are needed to convert the order and orientation from D virilis to D melanogaster! Slawson et. al., 2006 Genome Biology, 7(2):R15. Copyright © 2011, Washington University in St. Louis

“Wanderer” genes move between the dot chromosome and a
euchromatic site in the long arms; they adopt the properties (gene size, codon bias) of their local environment CG5262 CG9935 CG5367 rho-5 CG1732 dot Source: Leung et al (2010) Genetics 185: 1519 – 1534. Slide: Wilson Leung Notes: Careful annotation has convinced us that there are eight genes that are in a euchromatic domain (in the other chromosome arms) in one species, and in a heterochromatic domain (on the fourth) in the other species. (Some other candidates were discarded as being transposable elements, not true genes.) These genes adopt the properties of the surrounding domain – i.e., they are larger and show less codon bias when they are on the fourth chromosome, indicating that these properties are a consequence of being in that domain, not a property of the gene per se. We hope to identify and analyze more such genes in our comparative studies. Because we have restriction maps that confirm the fosmid assemblies, we know that these genes genuinely moved – but how is a mystery! CG11077 CG4038 CG11076 dot: D. virilis dot: D. melanogaster Leung et al 2010

But remember - what determines phenotypes. It’s not just your DNA…
But remember - what determines phenotypes? It’s not just your DNA….it’s how it is packaged! Environment (diet) (grey bars = folate) Phenotype Epigenetics ? Genotype Slide: Nicole Riddle, Washington University in St Louis. Source: RA Waterland, RL Jirtle (2003) Transposable elements: targets for early nutritional effects on epigenetic gene regulation. Mol Cell Biol 23: 5293 – 300. Notes: Environment can have a big impact on phenotype. Example: phenylketonurea - metabolic, genetic disorder leads to severe developmental phenotypes, retardation etc, but if the environment, ie diet is modified, no symptoms are observed. Data is shown here for the agouti mouse – the yellow coat color is due to an active transposable element (TE) driving a transcription factor in an incorrect tissue; if you feed a methyl donor (ie folate) to the mouse and you can shift the phenotype back to wildtype (brown). Development (Waterland and Jirtle 2003)

Higher resolution mapping: Chromatin ImmunoPrecipitation – ChIP (cells or nuclei)
1. Crosslink proteins to DNA 2. Isolate chromatin and sonicate qPCR 3. Incubate with antibody ChIP-chip* Slide: Nicole Riddle Notes: More recently we have looked at genome-wide patterns of histone modification using ChIP (chromatin immunoprecipitation) experiments. The chromatin is cross-linked (generally using formaldehyde), and then sheared into small fragments of a few hundred base pairs. The fragments are incubated with an antibody specific for a particular histone modification (for example, H3K9me3) or chromosomal protein; all of the chromatin fragments that bind the antibody are then collected. The cross-linking is reversed, and the selected population of DNA identified, either by qPCR (to look at a particular locus), or by using an oligonucleotide array to look at the whole unique genome, or by re-sequencing the DNA, and the identified population of DNA fragments is mapped back onto the sequenced genome. The resulting map shows the distribution of the particular histone modification or chromosomal protein. The modENCODE project ( has mapped numerous histone modifications and chromosomal proteins across the genome of D. melanogaster. 4. Isolate AB/chromatin complexes ChIP-seq 5. Isolate DNA from complexes

Mapping chromosomal proteins & histone modifications by ChIP-chip: chromosome arm 3L shows a distinct shift between heterochromatin and euchromatin. Centromere S2 cells Euchromatin Heterochromatin HP1a Su(var)3-9 H3K9me2 H3K9me3 genes Enrichment (log intensity ratio values) Slide: Nicole Riddle Source: Data discussed in Riddle et al (2011) Genome Res 21: 147 – 63 Notes: The ChIP data is presented here in a “wiggle” graph indicating enrichment; significant enrichment is indicated by the solid pink bar. This can be related to the gene map on the bottom line (genes in green). Here we are looking at the region at the base of chromosome 3L, where we see a transition from the euchromatic domain (left) to the pericentric heterochromatin (right). One sees a consistent shift to enrichment for the histone modifications (H3K9me2/3) and chromosomal proteins (HP1a and Su(var)3-9, an H3K9 histone methyl transferase) that we have found associated with heterochromatin formation and silencing. Euchromatin / heterochromatin transition point from Flybase. Pink boxes show significant enrichment (0.1% false discovery rate).

An expanded view of the fourth chromosome reveals
TSS (state 1, red) and Pc (state 6, grey) domains interspersed within heterochromatin (states 7 & 8, blue). Pericentric heterochromatin 10 Mb chr3L chr4 500 kb Red Variegating Slide: Wilson Leung Source: mapped according to the 9 state model of Kharchenko et al (2011). Notes: Using the maps of protein/histone modification data, we can identify 9 different chromatin “states”; for example, state 1, found at Transcription Start Sites is enriched for H3K4me3 and RNA polymerase. We can now map the different chromatin states across an expanded fourth chromosome. We find that while much of the chromosome is in states 7 and 8 (blue and light blue, associated with H3K9me2/3), there are domains in state 1 (red, associated with H3K4me2/3, transcription start sites) and state 6 (grey, associated with H3K27me3, regulated by the Polycomb system). Our variegating reporters lie in domains that are in state 1 or state 7/8 in Bg3 cells; note that the reporters showing full expression lie in domains that are found in state 6 in at least one cell type, indicating that this state is permissive for gene expression. BG3 cells, chromatin states: 1 2 3 4 5 6 7 8 9

Future: try to determine what feature drives 4th chromosome
Most 4th chromosome genes lie in heterochromatic space (blue), but active genes achieve state 1 (red) at the TSS Source: modENCODE data assembled in a local UCSC-style browser by Wilson Leung. Notes: We conclude that fourth chromosome genes have a special mechanism to block heterochromatin spreading across the promoters (TSS) of the active genes, or to displace such marks as part of the activation process. Note that a typical TSS chromatin has been assembled here, even though a 1360 repetitious element, a likely target for silencing, is nearby It will be interesting to see whether these genes have a special TSS motif not found in genes normally resident in euchromatin. 1360 repeat Future: try to determine what feature drives 4th chromosome gene expression that is absent from euchromatic genes (hsp70).

1 2 Heterochromatin formation on the dot chromosome…
Heterochromatin formation changes chromatin at the nucleosome level, eliminating HS sites at the TSS of euchromatic genes; silencing is dependent on HP1a 1 2 Fourth chromosome genes are larger, have more introns, and less codon bias than euchromatic genes Fourth chromosome genes show high levels of HP1a and H3K9 methylation over the body of the gene, but maintain access at the TSS. Next steps: what makes fourth chromosome genes robust? Lets look for fourth chromosome motifs! Slide: SCR Elgin

Eight new Drosophila genomes chosen at an evolutionary distance to facilitate motif finding
Source: Thomas Kaufman, Indiana University. Notes: Eight additional Drosophila species have been sequenced as part of the modENCODE project. These species were selected to be an appropriate evolutionary distance from D. melanogaster to facilitate motif hunting. Interestingly, some of these species appear to have an expanded dot chromosome, and some a rather small dot chromosome. The GEP plans to annotate the dot chromosomes from 2-3 of these species in order to search for TSS motifs specific to fourth chromosome genes. Expanded dot chromosomes?

Questions?

June 2008 Results using finishing/annotation in semester lab course -112 hr Quiz scores: precourse 12.8, postcourse 15.6. GEP school SURE 09 Bio Sample size: WU ~45, SURE , soph lab 90, all GEP Conclude that 1 semester is enough, 10 hr not enough. Instructors goal – use annotation to teach gene structure, any understanding of research is bonus! Test school All GEP 09-10 Results using annotation in genetics lab – 10 hr Quiz scores: precourse 4.0, postcourse 9.7 Copyright © 2008, Washington University in St Louis

Bioinformatics is a cost-effective research area for undergraduates
Genomics Education Partnership, Alumni Workshop June 2008 Bioinformatics is a cost-effective research area for undergraduates GCAT - Chip, SEEK, SynBio: Malcolm Campbell, Davidson College Teaching Genomics Consortium: Susan Singer, Carleton, Lois Banta, Williams (on-line curriculum) Aiptasia & Chamaecrista Genomics Explorers: Susan Singer, Carleton and Jodi Schwarz, Vassar _genomics/aiptasia Dynamic Gene, CSHL Dolan Center: Dave Miklos (annotating rice) iPLANT: DNA Subway, with CSHL Dolan Center (annotating Arabidopsis) Human Microbiome Project: Anne Rosenwald, Georgetown, Jena Canfield, Simmons, JC Ventor Institute Instructor: Professor Sarah CR Elgin Slide: Sources: Notes: Copyright © 2008, Washington University in St Louis

Bioinformatics is a cost-effective research area for undergraduates
Genomics Education Partnership, Alumni Workshop June 2008 Bioinformatics is a cost-effective research area for undergraduates JGI programs led by Cheryl Kerfeld, joint with ASM and Brad Goodner, Hiram College Undergraduate Research in Microbial Genome Analysis Tools for undergraduates to annotate microbial genomes as part of the GEBA Undergraduate Research in Microbial Functional Genomics Microbial work lends itself to vertical integration Other projects: bar coding for environmental studies regulatory and metabolic pathways protein folding (Foldit game) Instructor: Professor Sarah CR Elgin Slide: Sources: Notes: Copyright © 2008, Washington University in St Louis

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