Chapter 7 genome structure, chromatin, and the nucleosome (1)


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  • Comparative genomic organizations of the MHC class II region in mouse, domestic cat, and human. Brackets (A–C) indicate gene segments in human, but absent in mouse and cat. (D) A human gene segment absent in cat. (E) A mouse gene segment absent in human and cat. (F) A mouse segment absent in cat.
  • Five percent are genes that do stuff
  • Intra/inter- chormosomal rearrangement/duplications
  • Huge portion of your genome is just repetitive (junk DNA, dark genome)
  • Lengths of repeats could causesphenotypical adaptations
  • many of these retroviruses are linked back to diseasesRNA dependent-DNA reverse transcriptaseCan disrupt gene expression very far from where they are located on genomeThere are some inheritance factors in play here different results from mom or dad inheritanceThe sequences are located in genome and make up about 8%
  • 11% of genome is an Alu elementHas its own mechanism of replication Almost every chromosome has an Alu element in itNot sure why there, thought to be just junk DNA
  • BRCA1 is made 41.5% by alu elementsThis could be important in regarding the diseases that come from BRCA1 (breast cancer) can cause recombination to occur where it isnt supposed to Alu elements are recombinational “hot spots” in certain genes that can cause issuesAlu elements tends to generally produce a negative impact
  • We know that we need vitamin C but we can’t make itSome primates can make it on their own We are in the suborder of the right, so we inherited not being able to GULO is an enzyme that does it, but humans do not have it functioning rightAlu elements could be at fault for this
  • Retrocyclin production is found in us but it is turned offIt is an HIV resistant gene that they are trying to turn back on to fight off the virus
  • Mitochondria have their own genome and produce things that our nuclear genomes do notWe make some stuff, they make other stuff (horizontal gene transfer occurs)Arabidopsis: new Mendelian pea plantCancer: you will see lots of horizontal gene transfer happening here
  • Eats DNA and produces new enzymes from itEating it and using it as its ownDoesn’t need to undergo recombination that way
  • While we destroy it, this actively uses the DNA for itself
  • RNA structure is more complex than it looks
  • Entire new topic based around the fact that the whole genome is being transcribed into RNA (conductone)Nematode is almost identical to humans in terms of genes, its made only of 1000 cells33/93% of genome is transcribed (wide generalization but still shows how conservation is prevalent)RNA is showing up to be more important than proteins
  • Long noncoding RNA can have various functions Junk Byproduct Scaffold Guide Effector Enhancer or activator
  • RNA polymerase is often found in antisense locations
  • MicroRNA are being found everywhere in almost every disease and all around the bodyMicroRNA can be used for shutting off a process very quickly Glucose/Lactose exampleThe food we eat could have effects on it through MicroRNA
  • Brain - tissue specificHas been linked to diabetes, some cancers, can be found in ears, eyes, heartViruses could use this to regulate their host
  • Can use microRNAs to go about silencing certain genesUses same Dicer/RISC process
  • RNA may be a way to pass on genes outside of the genomeKnocked out RNA processing machinery but for some reason the defense provided by the RNA somehow remained in subsequent generationsLamarckism- it is possible that what you do with your body does have an impact on genes and thusly offspring
  • Chromosomes have centromeres- only one per chromosome
  • 46 total chromosomes: 22 pairs of autosomal chromosomes, 1 pair of sex chromosomesSister Chromatids- different strands of DNA, not linked by phosphate backboneTwo sister chormatids make one duplicated chromosomesTwo chromosomes make one pair of homologous chromosomes TED Talk: Animations of unseen biology:Shows the lining up of chromosomes on metaphase plate, then when they split up and go to each poleCentromere is the interface b/w the microtubules and the chromosomes it is made up of thousands of proteins
  • If no centromere then the chromosomes wont be pulled apartIf more than one centromere then the chromosome would shatter because pulled apart too much
  • TopLeft: chromosomes lined up on metaphase plate
  • Telomeres are at the ends of the chromosomes and haven’t been discovered completely
  • Cell cycle from general bioMost working cells are just in G0 phase (quiescent) They go on to dividing only if they need to do soS phase: 4 copies of genes because of duplicationSpindle fibers are microtubules There are checkpoint between each phase
  • Showed animation of the processChromosome replication is in S phaseChromosome segregation is in M phaseDuring G2 the sister chromatids are kept together by cohesinG1,S,G2 is in interphaseMitosis Prophase: Metaphase: bivalent attachment occurs along the center plate anaphase: chromosomes are pulled apart and cohesin is losttelophase:
  • Usually in other processes that are not mitosis, the chromosomes are free Only very compact structure during Mitosis DNA is very lose during interphase (10nm)DNA is very condensed during mitosis (30nm)
  • Gap phases are the checkpoints that occur in the cycle they stop when they need to fix damages that are present they stop to keep growing and making sure there is enough nutrients to keep goingCyclins and cyclin-dependent-kinases (CDKs) are involved in cell cycle somehowRB proteins need to be phosphorylated for cycle to keep going when phosphoylated the RB is inhibited and cycle goes on
  • KNOW THIS WHOLE THINGBe able to explain little parts of it, might be a picture on the test ATM/R can shut down cell cycle if it senses stressWhen you poly-ubiquitinatesecurin, you are degrading it, which allows you to continue through anaphase
  • ATM can respond to damaged DNA via many pathways Ex: it can activate BRCA1 which works for DNA repair Ex: can activate P53 which can stop topoII which stops DNA replication
  • Prophase: condensin-condenses DNA to make it the condensed DNA that we see in mitosiscohesin-Metaphase: is the alignment that occurs along the center, it breaks down after tension rises there are proteins that make sure that everything lines up accordinglyAnaphase: cohesin gets degradedTelophase: condensin gets degraded
  • Cohesin is added during replication and needed for proper metaphase alignmentCohesin mutations almost never are seen because people wouldn’t survive with it
  • Aurora can be in different states when there is no tension then there is an access point on the aurora and phosphorylation can happen when tension occurs then there is no access to it and phosphorylation cant occurLining up along the metaphase plate occurs correctly because the length of the microtubules connected to the chromosomes when there are issues in the tension, proteins manipulate the chromosome so it falls into place in the center of the plate
  • DNA is physically linked over to the kinetochore (the microtubules) via small proteins all these phosphorylations are part of the process that allow for DNA to attach to microtubule tension assists in the process as well
  • Process of adding the cohesins during the interphase part (S phase)
  • Cohesin and condensin are the key proteins involved in the process keep things together or condense them downMade up of SMC
  • Cohesin chopped up by protease and spindles pull apartThere are spindle assembly proteins that make sure all the tension is where it needs to be for the process to continue
  • Cohesin physically links two pieces of DNA that are not attached (during Sphase) it is removed during metaphase-anaphase swap point Condensins are added on during prophase they take one piece of DNA and they make it come closer to itself it is removed at the end (telophase)
  • Separase cleaves the chromosomes cohesinand allows them to come apart
  • Fig. 4. Comparison of models to raw images of folded chromatin. (A)Electron cryo-microscopy images of a folded chromatin array containing 22nucleosomes with a repeat length of 177 bp. The particles are visualizedunperturbed in the frozen hydrated state and oriented randomly in icecontaining 1.0 mM Mg2. The particles in each row represent similar views.(Scale bar: 100 nm.) (B) Views of the interdigitated one-start helix model (Fig.3A) that closely match the EM images from the corresponding row in A. (C)Views of the adapted two-start crossed-linker model (Fig. 3B) that are equivalentto those of the interdigitated model from the corresponding row in B.The varying orientation of the coordinate axes describes the threedimensionalorientation of the models in the corresponding rows.
  • Genomic Architecture by Courtney Humphries CommentPDFPhotograph by Stu RosnerIf it were not for an elegant design, your DNA might be a tangle of crossed lines and knots. Graduate student Erez Lieberman-Aiden was part of a team that discovered how the genome packs information accessibly into a tiny ball of hierarchical folds.KeywordsBroad Institute, DNA, genomics, systems biologyOne of the enduring puzzles of biology is how our large, complex genomes function, let alone fit, inside the cramped compartment of a cell’s nucleus. The total DNA in a human cell—roughly two meters in length—is somehow packed into an organelle just a hundredth of a millimeter in diameter.  Even more impressive, the cell is able to locate, access, and transcribe genes within this dense bundle. Now a study published in Science describes how researchers at Harvard and the University of Massachusetts Memorial Medical Center used a new technique to create a three-dimensional map of the human genome, revealing how DNA pulls off this stunning feat of organization. Erez Lieberman-Aiden, a Harvard graduate student and co-first author of the paper, says, “The challenge we face with the genome is that it occupies a nether region where a lot of our technologies don’t work very well.” At the smallest scale, scientists know that human DNA forms a double helix, and that this helix is wound around proteins to form condensed bundles. On a larger scale, we know that long stretches of bundled DNA are divided into individual chromosomes that are visible under a microscope only occasionally, when they compact into a classic X-like structure during cell division. The area between these two scales has been difficult to study with existing techniques.Eric Lander, professor of systems biology and director of the Broad Institute of Harvard and MIT, partnered with Job Dekker, associate professor of biochemistry and molecular pharmacology at UMass Medical School to solve the problem. Postdoctoral fellow and co-first author Nynke van Berkum of UMass and Lieberman-Aiden led the development of a technique called Hi-C, adapted from technology previously developed in Dekker’s lab. It uses formaldehyde to “freeze” the position of DNA in the nucleus by gluing together DNA strands that are near one another. The DNA is then broken into many pieces, which are then sequenced to reveal the identities of DNA sequences close to one another in space. The process is repeated for millions of cells and the results averaged to create a spatial, three-dimensional model of DNA in cells.The researchers discovered a feature about the overall organization of DNA that helps explain how individual genes are accessed. DNA is grouped into two compartments in the nucleus: one, loosely packed, that contains active genes; another, densely packed, where inactive genes lie. It’s been known that DNA unwinds somewhat to make genes accessible, but the new study suggests that DNA actively snakes its way to different parts of the nucleus depending on whether genes are needed or not. Lieberman-Aiden compares the system to a work space: the genes that are active are like files loosely spread out on a desk; unneeded genes are like files packed and stored in a file cabinet.Leonid A. Mirny and Erez Lieberman-AidenIn addition, the team discovered how DNA manages to compact itself into the nucleus. Lieberman-Aiden was looking for a physical model that could explain the group’s data when he found the answer: in a 1988 physics paper predicting that a molecule similar to DNA could form a structure resembling a Peano curve—a  fractal design discovered in 1890 by an Italian mathematician that allows a continuous curve to densely fill a space without ever crossing itself (see the image right).DNA, it turns out, crumples into a hierarchical series of folds that enable it to pack densely without forming knots or tangles. The researchers call this structure a fractal globule. And Lieberman-Aiden says it makes perfect sense as a way to manage the information in the genome. “If I was trying to build a library, I’d want the volumes to be compact in one place, I’d want them to be organized, and I’d want them to be accessible,” he explains. A fractal globule allows the genome to follow these same principles. Although the current study provides a fundamental insight into genome organization, Dekker says the “resolution of the current spatial map we’ve built is not high enough.” Right now, the map is at the level of a single megabase, or one million base pairs—the equivalent of breaking the entire genome into 3,000 pieces. It will require many more iterations of the technique to be able to resolve structure on a smaller level.  Lieberman-Aiden says other scientists “have realized that this type of approach to studying the three-dimensional structure of the genome is really quite transformative.” An improved three-dimensional map of DNA’s structure could help answer questions about how genes work that aren’t apparent in the genome sequence—for instance, how genes are controlled by other DNA sequences in the genome called regulatory elements. These stretches of DNA often are not adjacent to their target genes in sequence, but they may turn out to be close together in physical space. The researchers also plan to study how the DNA map varies among cells from different species and among different cell types. The mapping technique could even offer new insights, they believe, into differences between cells that are healthy and those that are diseased.
  • Three possibilities for the distribution of parental histones are presented. In each case, the parental mark is recognized by a chromatin-binding protein, or reader protein, that in turn recruits a chromatin modifier, or writer protein. a | Random histone distribution. Parental histone H3 and H4 with marks (unsplit or reassociated dimers) are distributed randomly onto daughter strands and chromatin density is restored by the deposition of new H3–H4 dimers. To avoid the dilution of histone marks, active maintenance requires first a deacetylation step, which involves a histone deacetylase (HDAC), followed by histone modification that is guided by neighbouring parental nucleosomes (an interparticle process). b | Semi-conservative histone distribution. Parental dimers with marks segregate evenly onto each daughter strand and nucleosomes are completed by the deposition of new H3–H4 dimers. After deacetylation, 'hemimodified' nucleosomes provide a template for the transmission of parental marks to newly deposited H3–H4 dimers (an intraparticle process). c | Asymmetric histone distribution. Parental H3–H4 dimers with marks are redistributed onto daughter strands in an asymmetric manner. This is possibly dictated by the intrinsic strand bias that is introduced during DNA replication, and induces a switch from one chromatin state to another. The maintenance of histone modifications requires interstrand crosstalk.
  • Chapter 7 genome structure, chromatin, and the nucleosome (1)

    1. 1. Chapter 7 Genome Structure, Chromatin, and the Nucleosome
    2. 2. Chapter Overview I. Chromosome & Genome Overview II. Overall Genome Organization III. Introns and Intergenic DNA IV. RNA V. Chromosome Components VI. The Cell Cycle: Chromosome Duplication and Segregation VII. The Nucleosome VIII. Chromatin Structure IX. Regulation of Chromatin Structure
    3. 3. Chromosome & Genome Overview • Chromosome – one molecule of DNA and associated proteins (half of the mass) • Chromatin – complex of DNA and proteins that make up the chromosome – DNA – Nucleosome (histones) – Non-histone proteins – transcription, replication, repair, recombination, topology • Allows 2 meters of DNA to fit in the cell • Stability/protection from degradation • Means of transmission to daughter cells • Overall architecture contributes to regulation of expression, recombination I.
    4. 4. Prokaryotes • Millions of base pairs • 1 mm chromosome into 1 m • Usually circular (although linear has been found) • Have proteins similar to histones • Must be separated by topoisomerase after replication • Usually 1 complete copy of the chromosome in nucleoid • Plasmids Ruptured E. coli
    5. 5. Eukaryotes • Billions of base pairs (compact, restricted access) • 2 to 50 chromosomes • Usually diploid with 2 homologs (thousands in protozoa Tetrahymena) • Haploid • Polyploid – Allows for more RNA (and protein) generation – Megakaryocytes (~32-64 copies of each chromosome) for platelet production
    6. 6. Humans • Autosomes – 22 pairs, 1 copy from each parent • Sex chromosomes (X and Y) – carry sex-determining genes. Evolved ~200-300 million years ago after split from monotremes, evolved independently in reptiles, birds, plants • Mitochondrial DNA – mostly from mother • Thousands of microorganisms – from mother, environment
    7. 7. Genome size roughly correlated with an organism’s complexity, number of genes more closely linked to complexity
    8. 8. Genome Density • Viruses – some use both strands and have overlapping genes • E. coli genome – composed almost entirely of genes, with only a few transcriptional regulatory regions, and operons to control many genes • More complex organisms have decreased gene density – Intergenic sequences • Unique – mutated genes or pseudogenes • Repetitive • Junk DNA – Regulatory sequences – Introns – Still thousands of overlapping genes
    9. 9.
    10. 10. Veeramachaneni et. al., 2004 MutY Homolog Target of Early Growth Response Factor 1 Testis- specific kinase 2 Chromosome 1, P arm
    11. 11. Mitochondria “stop” (TAA) only after polyA tail is added
    12. 12. Overall Genome OrganizationII. • Very similar • Chimpanzees, gorillas, orangutans – 24 chromosomes – MRCA 14 mya • Humans – 23 chromosomes – Chromosome 2 is a fusion of two other chromosomes, homologues of which are found in other hominidae – Vestigial centromere and telomeres – In addition, ~2-6% Neanderthal DNA
    13. 13. Genome Organization – Humans vs. Mice Cut human genome into ~150 pieces to piece together the mouse genome. Location usually irrelevant, but clustering is often required. Example: “On chromosome 11, there are five functional and two nonfunctional beta globin genes in a row. If the beta globin genes are removed from their surroundings, they are not properly regulated. Also, if you mix up the order of the genes, they are expressed at the wrong times.” --Dr. Barry Starr, Stanford University
    14. 14.
    15. 15. New Genes on Chromosome 2 at Fusion Site • 15,000 new base pairs, seem to have come from Chromosome 9 prior to fusion event Transcription control, growth/development Non-functional Unknown function Bacterial protein that synthesizes B12 (brain development?)
    16. 16. The 44 Chromosome Man • Chromosome 14 and 15 fused • Perfectly normal • His children (with 46 female) would carry 1 copy of this fusion, and 2 copies with a 44 female
    17. 17. Y Chromosome Evolution
    18. 18. • Pseudoautosomal regions (PAR) – homologous to X chromosome, recombine during meiosis • X-transposed – 3-4 million years ago in humans, contains 2 genes • X-degenerate – 16 housekeeping genes, homologs on X, similar across primates • Ampliconic – 60 male-specific genes, repetitive, palindromes (for repair) • Y chromosome has been replaced in mice: – SRY – male-specific transcription factor – Eif2s3y – spermatogonial proliferation factor
    19. 19. Extensive Adaptive Evolution Specifically Targeting the X Chromosome of Chimpanzees • Genetic mutations that boost an individual's adaptability have greater chances of getting through to X chromosomes, whereas only a few adaptations on the autosomes have occurred (most of which are related to immunity gene clusters) • A new beneficial variant on one X chromosome in the female can 'hide itself' if it is not expressed as strongly as the old variant sitting on the other copy of the X chromosome (i.e. recessive). A new beneficial recessive variant does not immediately provide a benefit for the females. On the other hand, the males only have one X chromosome and it is expressed immediately, thus enabling natural selection to 'catch sight' of it. This does not apply to the autosomes • 30% of amino acid substitutions on the X chromosome since humans and chimpanzees diverged (4-6 mya) were adaptive/beneficial for the chimpanzee. • Overall the X chromosome is less variable than the autosomes between humans and chimps because natural selection works stronger on the X chromosome since variations have a harder time “hiding” • Y chromosome is fastest evolving
    20. 20. X Inactivation A mouse’s retinas Mouse brain cornea, skin, cartilage and inner ear
    21. 21. How We Became Human • 98-99% identical (1.23% different, ~35 million), most divergence is in Y chromosome • ~95% identical when insertions and deletions are considered (~ 5 million) • 29% identical proteins, most differ by only two amino acids on average (hemoglobin only differs by a single amino acid) • 1,576 apparent inversions between the chimp and human genomes; more than half occurred sometime during human evolution • ~580 of 25,000 common genes seem to have been positively selected for in humans • ~1418 gene differences (689 human, 729 chimps) that were lost in each species, 30 million SNPs • Chimps have more variation (even between siblings), rhesus macaque has three times as much genetic variation as humans • Up to 40% difference in protein expression levels,9171,1541283,00.html
    22. 22. Humans vs. Primates • Tiny differences, sprinkled throughout the genome, have made all the difference. Agriculture, language, art, music, technology and philosophy are somehow encoded within minute fractions of our genetic code. They give us the ability to speak and write and read, to compose symphonies, paint masterpieces and delve into the molecular biology that makes us what we are. • FOXP2 – reading/writing (humans with defect have difficulty with both), evolved within past 200,000 years. Differs in only 2 of 715 amino acids from chimps. • MYH16 – myosin variant in jaw muscles, found in all primates but humans. Allowed for the evolution of smaller jaw muscles 2 million years ago, and allowed the braincase and brain to grow larger. • Protein (domain) DUF1220 – found in areas of the brain associated with higher cognitive function. 212 copies in humans, 37 or less in primates, 1 in mice/rats • HACNS1 (enhancer) – 13 nucleotide difference between humans and chimps (many more changes than expected via random drift). The human, chimp, and macaque gene was inserted into mice. It was active in the hands, feet, and throat. Human version showed most activity • Yet, only 19,000 genes, so likely the noncoding regions also play an important role in distinguishing humans from other primates, and affect expression/regulation,9171,1541283,00.html Video: Humans and chimps:
    23. 23. 48/3200 = 1.5% III. ENCODE project (2012) showed 80% of genome biochemically active, containing many gene switches and regulatory elements
    24. 24. Introns and Intergenic DNA • Most of the genome is non-coding • Introns increase with complexity (S. cerevisiae only has introns in 3.5% of genes) • Average transcribed region is 27 kb, average gene is 1.3 kb (~5%) • Vinculin: 22 exons over 75,000 base pairs, 1066 amino acids (4.2%) • Introns – Make coding sequences discontinuous – Removed by splicing – Allow for diversification – Ribozymes or miRNA?
    25. 25. Alternative SplicingExons and Domains
    26. 26. Unique Intergenic DNA • ~25% of genome – Regulatory – Gene fragments – Nonfunctional mutant genes – Pseudogenes (not expressed, no regulatory region) • Gene duplications (~5% of genome) • Unequal crossing over • Transposons, retrotransposons – 40-50%, most unable to transpose • Reverse transcriptase (from a virus) incorporating random mRNA into genome • Nearly complete viral genomes – 1.3% • Example: ~80 genes on Y chromosome, but 282 pseudogenes • Many pseudogenes are shut down via methylation • Some may encode proteins linked to cancer – miRNA (Section IV) – Origins of replication, segregation, telomeres (Section V) Repeats, transposons Unique, transposons Genes Gene- related 5% 20% 25% 50%
    27. 27. AAAA AAAA
    28. 28. Unequal Crossing Over
    29. 29. Gene Duplications on Chromosome 16 Red – interchromosomal Blue - intrachromosomal
    30. 30. Repetitive Intergenic DNA • ~50% of the human genomic DNA is repetitive – Microsatellite • Repeating units 13 bp, tandem repeats, • Most common is dinucleotide repeat (3% of genome) like CACACA… • Arise from mistakes in DNA synthesis – Genome-wide repeats • >100 bp, some over 1 kb, dispersed or clustered • Rare, but over time have reached 45% of genome • Transposable elements (retroelements) – move to new positions, often leaving original copy behind, such as Alu, LINE-1 (can silence nearby genes, leading to cancer, schizophrenia), SVA elements which are now 33% of genome • Major role in evolution and disease • Repetitive DNA exists in E. coli, but far less common – Repair systems – Gene disruption due to lack of introns – Not as competitive with more DNA to copy Repeats, transposons Unique, transposons Genes Gene- related 5% 20% 25% 50%
    31. 31. Repetitive/Intergenic DNA Function • Junk DNA? Or DNA with an unknown function? • Regulation, genetic variation • Onions have 12X the DNA of humans, retain more DNA than lose it • Comparison of fruit flies vs crickets (11X more DNA) showed crickets lose DNA much more slowly, and have more variability • Although there is a plant that has rid itself of nearly all junk DNA – 97% of its 80 mbp encodes proteins • Stable maintenance of these sequences over hundreds of thousands of generations suggest that intergenic DNA confers a selective advantage • 80% of human/mice homology is not in proteins, but in noncoding DNA • Repetitive DNA: – Different lengths of repeats sequences in dogs correlate with morphological differences in the dogs' skulls and limbs, 51 regions of the dog genome associated with phenotypic variation among breeds in 57 traits – Link between vasopressin receptor gene (associated with pair-bonding) and repeat length • Repeat expansion and contraction is a very useful mechanism for imposing rapid evolution. Tandem repeat units can be added or subtracted by slipped-strand mispairing during DNA replication. This type of mutation happens at least 100,000 times as often as simple point mutations. Repeat length changes can make subtle alterations to proteins, whereas point mutations are usually either neutral or fatal to the protein Fondon, Garner, PNAS 2004
    32. 32. Endogenous Retroviruses • ERVs, a repeat derived from ancient viral infections • 8% of total genome (98,000 fragments) • Neanderthal and Denisovan ERVs found in modern humans • Linked to diseases in humans such as MS, possibly schizophrenia • Mouse study of ERVs: – ERVs significantly disrupt gene expression, up to 12.5 kb away from transcription site – 100 genes were found to be disrupted via premature polyadenylation, up to 50-fold change in expression – A mouse gene containing an ERV inherited from the father produced only an incomplete, truncated form of messenger RNA (mRNA); if the ERV came from the mother, both the truncated transcript and nearly normal levels of the full-length mRNA were produced from the gene – Can cause biological variation and diversity
    33. 33. Alu Element • Descended from signal recognition particle which targets proteins to the ER • 300 bp, 1 million copies, ~11% of genome
    34. 34. 41.5% of BRCA1 intronic DNA is Alu elements (138 copies) “Alu sequences have often been regarded as genomic instability factors because they are responsible for recombinational "hot spots" in certain genes and are frequently involved in exon shuffling during meiosis as a result of non- homologous recombination. These sequences may also act as regulatory factors in transcription, with structural roles (as "physical separators" of protein-protein interactions during chromosome condensation in cellular division) and functional roles (in alternative "splicing" or as a connection between transcription factors) are being proposed.” “BRCA1 exon 5-7 deletion described in German families results from a non-allelic homologous recombination between AluSx in intron 3 and AluSc in intron 7. Both Alu repeats share a homologous region of 15 bp at the crossover site. (Preisler-Adams et al., 2006)”
    35. 35. Selfish Genes Disrupt Segregation • Segregation disorder (Drosophila) – 99% inheritance • Sd produces a truncated version of the RanGAP nuclear transport protein, and its presence interferes with the normal processing of Rsps-bearing sperm
    36. 36. Why Can’t Humans Synthesize Vitamin C? • 500 million year old process, scavenges reactive oxygen • Most plants and animals synthesize vitamin C from glucose • L-Gulono-gamma-lactone oxidase (GULO), which catalyzes the last step of biosynthesis, missing in humans • Functional rat GULO has 12 exons, the human pseudogene on chromosome 8 only has 5 exons • The human gene has a deletion of a single amino acid, several mutations, two stop codons, two single nucleotide deletions, and an insertion • In addition there are two Alu sequences inserted in the vicinity of a presumed position of lost exon 11 during the same period as GULO lost its function Primates: Suborder Strepsirrhines Suborder Haplorhini Split 63 mya
    37. 37. A Drug To Re-Awaken Ancient Human Genes And Fight HIV • Dr. Alex Cole’s lab, UCF • "Junk DNA" are inactive parts of your genome, switched off long ago in evolutionary history. Now scientists say there's a junk gene that fights HIV. And they've discovered how to turn it back on. • They have re-awakened the human genome's latent potential to make us all into HIV-resistant creatures. • Old World monkeys had a built-in immunity to HIV: a protein called retrocyclin (18 amino acids, only circular proteins in body), which can prevent HIV from entering cells and starting an infection. • Humans have the gene but it contains a nonsense mutation that had turned it off • Dr. Cole’s lab used aminoglycosides to read-through the premature termination codon found in the mRNA transcripts and therefore start making retrocyclin again • In preliminary tests the human cells made retrocyclin, fended off HIV, and effectively became AIDS-resistant. And it was done entirely using the latent potential in the so-called junk DNA of the human genome.
    38. 38. Horizontal Gene Transfer and Genomic Duplications • Transformation, transduction, conjugation • Bacterial resistance to antibiotics • Mitochondria/chloroplast DNA into plant/human genomes • Millions of years ago, a cluster of 23 genes jumped from one strain of mold commonly found on starchy foods like bread and potatoes, Aspergillus, to another strain of mold that lives in herbivore dung and specializes in breaking down plant fibers, Podospora, which encodes a toxic compound sterigmatocystin (for protection) • Arabidopsis (which replaces pea plants for genetic studies) duplicated its entire genome 38 million years ago, about 1/3rd of duplications remain intact • 11% of Neisseria gonorrhoeae have human LINE-1 retrotransposon DNA • Cancer
    39. 39. Animal Eats DNA to Obtain New Enzymes • bdelloid rotifer – found in lakes, can survive many years without water, tolerate high levels of radiation • Ingests DNA from environment • Up to 10 per cent of the active genes of an organism that has survived 80 million years without sex • Of ~29,000 matched transcripts, ~10% were inferred from blastx matches to be horizontally acquired, mainly from eubacteria but also from fungi, protists, and algae
    40. 40. Approximately 80% of horizontally acquired genes expressed in bdelloids code for enzymes, and these represent 39% of enzymes in identified pathways. Many enzymes encoded by foreign genes enhance biochemistry in bdelloids (toxin degradation or generation of antioxidants and key metabolites)
    41. 41. RNA • Coding: messenger RNA (mRNA) – 5’ UTR • 7methyl-G cap bound by cap binding proteins • Translation regulation – 3’ UTR • Stability elements • Subcellular localization (zip codes) • poly(A) tail • Non-coding RNAs: – Ribosomal RNA (rRNA) – Transfer RNA (tRNA) – Micro RNA (miRNA) – Short interfering RNA (siRNA) IV.
    42. 42. RNA Conductome (or Transcriptome) • Traditional view: only proteins are important, most of the genome is nonfunctional – 1.2% of genome encodes proteins – Huge portion of genome intergenic DNA (transposons, repeats) • Today it is known that RNA plays an important role – Nematode has 1000 cells, but encodes as many proteins (19,300) as humans – 3-8% of genome conserved between humans, mice, and dogs – 33% to 93%* of the genome is transcribed (*from an intensive 1% of genome study called ENCODE, which took 44 sections of genome of low/high gene density and low/high conservation to mice). Much of the genome is comprised of “gene switches” to turn genes on and off – 4520 of 158,807 mouse transcripts form antisense pairs with exons • MicroRNAs – small non-coding RNAs that regulate gene expression by interfering with mRNA function • 3000 ncRNAs found, predicted to be at least 12,000 – increase gene transcription • 400 long ncRNAs found in the livers of mice, which were found to prevent maturing red cell death (a step to leukemia) • RNA is involved in all aspects of regulation of cellular processes, including chromatin remodeling and epigenetic memory, transcription factor nuclear trafficking, and transcriptional activation or repression • dsRNA linked to heterochromatin silencing, keeping transposons in check • Millions of genes, as opposed to 20,000? Melissa Lee Phillips, John S. Mattick, The Scientist, October 2007
    43. 43. Long Noncoding RNA Signal for viral infection
    44. 44. Sense and Antisense RNA • Bidirectional transcription is more common than previously thought and has implications for understanding the complexity of gene regulation • RNA polymerase is often present at antisense location and transcription is initiated although not often elongated • Some genes may be self-regulated by antisense transcription • RNA polymerase may induce negative supercoiling upstream to help regulate transcription
    45. 45. MicroRNA / RNA Interference • Discovered in 1993 (lin-4) • ssRNA that is not translated, but forms a hairpin secondary structure • Originate from precursors, eventually processed to ~21 nt • Regulates post-transcriptional gene expression via down-regulation • Often not 100% complementary to the target • Important in development • Plant RNAs found in mammals: – 40 plant miRNAs found in blood – MIR168a (found at high levels in rice) binds to LDLRAP1 mRNA, reducing the protein levels and ultimately impairing the removal of LDL from the blood
    46. 46. MicroRNA • Tissue-specific. Involved in setting up the basic body plan, nervous system development, cholesterol levels, bone formation, wound healing, pituitary hormone secretion, heart attacks, cancer • Diabetes, cancer – miR-483-3p located in an intron within Insulin-like Growth Factor 2 (IGF2). Found elevated in liver, breast, colon cancer, and has oncogenic properties. Affected by maternal diet and suppresses Growth-Differentiation Factor 3 (GDF3), leading to weight gain • Brain – miRNAs inhibit certain protein synthesis, when a synaspe is activated by a thought/sound/etc., miRNA is degraded and protein synthesis strengthens the synapse, allowing memory formation • Ear – development, functioning. Conserved between humans and zebrafish • Eye – development. Flies missing Dicer have abnormally small eyes • Heart – Deletion of miR-1-2 causes heart defects, from deformation to electrical conduction to cell- cycle control • Immune system – miR-155 knockout has faulty B and T lymphocytes and dendritic cells. Viruses may also use miRNAs to regulate host genes (i.e. HSV-1 inhibiting apoptosis of infected neurons) • Blood cells – blood vessel formation by suppressing inhibitors. miR-15/16 found at chromosomal region deleted in over half of B cell chronic lymphocytic leukemias (B-CLL) • Insulin secretion (miR-375) • Muscle – miR-1 mutation produces abnormally muscular sheep • Liver – Hepatitis C binds to miR-122 to stabilize its own RNA and promote replication • Angiogenesis – miR-296 elevated in primary tumor endothelial cells isolated from human brain tumors. Directly targets the hepatocyte growth factor-regulated tyrosine kinase substrate (HGS) mRNA, leading to decreased levels of HGS and thereby reducing HGS-mediated degradation of the growth factor receptors VEGFR2 and PDGFRbeta • Atherosclerosis • p53 – regulated by 8 microRNAs in sperm production • Pancreatic cancer – 25 miRNAs expressed at different levels in 90% of samples • Polycystic Ovary Syndrome – over expression of miR-93 and decreased expression of GLUT4, a key protein that regulates fat's use of glucose for energy
    47. 47. siRNA • Small interfering RNA, similar to miRNA but originates from dsRNA • Degraded by Dicer, RISC (RNA inducing silencing complex) forms • Most commonly a response to foreign RNA (viruses, transposons) and is often 100% complementary to the target
    48. 48. RNA Interference • Roundworms infected with Flock House virus (only known infection) • Their progeny (who had RNAi machinery intentionally turned off) were exposed to the virus and still able to defend themselves for more than 100 generations (nearly a year) after the initial infection. • Extrachromosomal transmission • Lamarckism? Rechavi et. al, Cell 2011
    49. 49. Chromosome Components • Origin of replication – 30-40 kb apart (100k per chromosome) throughout entire chromosome in non-coding regions (Chapter 8) • Centromeres – only 1 per chromosome, ~40 kb of repeats (only 200 bp and non-repetitive in yeast) – Kinetochore (protein) attaches to it, links to microtubules • Telomeres – resistant to recombination and degradation (associated proteins) – Single-stranded, repeat (TTAGGG in humans) – Telomerase V.
    50. 50. 22 pairs of autosomal chromosomes 1 pair of sex chromosomes 46 total  p arm q arm Sister chromatids – different strands of DNA, not linked by phosphate backboneSister Chromatids and Homologous Chromosomes
    51. 51. The Centromere
    52. 52. (protein) (DNA) _of_unseeable_biology.html 4:45-9:00
    53. 53. The Telomere 3’ end can be thousands of bases long Telomeres (yellow) may anchor to cell edge during division
    54. 54. The Cell Cycle: Chromosome Duplication and Segregation • G0 – gap phase, resting (quiescent) • G1 – growth, preparation • S (synthesis) phase – synthesis, each chromosome is duplicated – 2 sets of sister chromatids – 2 chromosomes x 2 copies = 4 copies of a gene – Histone replication – Induced at G1/S transition, over a 25-fold mRNA increase in S phase • G2 – growth, preparation • M (mitosis) phase – Sister chromatids bound to mitotic spindle at kinetochore (centromere + protein complex) – Spindles are microtubules attached to centrosomes/pole bodies VI.
    55. 55. Animation 1 chromosome (1 piece of dsDNA)
    56. 56. More compact Beads (nucleosomes) on a string
    57. 57. Gap Phases • Cell cycle checkpoints • Cells prepare for next cell cycle stage • Cells check that previous stage is completed properly • Prior to entering S phase, a cell must reach a certain size and level of protein synthesis to ensure there will be enough proteins and nutrients to complete next cycle • Cells with DNA damage arrest in G1 before synthesis or G2 before mitosis to allow repair Movement through the cell cycle is driven by the activities of complexes of cyclins and cyclin-dependent kinases (CDKs), which phosphorylate retinoblastoma (RB)-family 'pocket proteins', thereby blocking their growth-inhibitory functions and permitting cell-cycle progression. Cyclins are shown as triangles.
    58. 58. Example: Protein signals prevent CDC20 from activating ubiquitin (which degrades proteins that are holding the cell at anaphase)
    59. 59. Mitosis • Maintains parental chromosome number • Prophase – Cohesin/condensin • Metaphase – Bivalent attachment causes alignment at metaphase plate, tension on chromosomes (essential to keep kinetochores attached) • Anaphase – Cohesin cleavage by separase • Telophase – Chromosomes decondense (loss of condensin) • Cytokinesis Ubiquitination and mitosis
    60. 60. Cohesin mutation
    61. 61. Proteins Involved in Chromosome Alignment Aurora B checks and regulates microtubule attachment, senses tension Dynein controls alignment
    62. 62.
    63. 63. S Phase 1 piece of dsDNA
    64. 64. Cell Cycle and Chromosome Duplication and Segregation • Chromosome duplication, condensation • Cohesion and condensation are mediated by SMC (structural maintenance of chromosome) proteins • Cohesin – two SMC + two non-SMC proteins, forming a ringed structure • Condensin – similar ring structure to cohesin • Mitotic spindles (microtubules) form, attach from centrosomes to kinetochore • Cohesin keeps tension on chromatids, prevents migrating towards poles • Cohesin cleaved, chromatids pull apart
    65. 65. Spindle assembly checkpoint proteins prevent premature segregation by binding to kinetochores that are not attached to spindles or under tension
    66. 66. Condensin/cohesin may be responsible for chromosome condensation
    67. 67. Other models
    68. 68. Meiosis • Two rounds of segregation, reduces parental number • Meiosis I – Monovalent attachment – both kinetochores of each sister chromatid are attached to the same microtubule spindle – Tension (essential) by crossing over (only 1 pair) – 30-40 crossovers. A sequencing study of 91 sperm showed ~23 recombinations per sperm – X and Y chromosomes cross over, and non-sex genes do swap (PAR), so females could have Y chromosome DNA, and males could have DNA from their father’s X chromosome • Meiosis II – Similar to mitosis without DNA replication – Most separase cleavage in anaphase II
    69. 69. chiasma 2N 4N 2N N Cohesin cleavage
    70. 70. Shugoshin recruits PP2A to centromeres where it locally dephosphorylates Rec8, rendering centromeric Rec8 resistant to separase
    71. 71. Cohesin cleavage
    72. 72. The Nucleosome • Nucleosomes are the building blocks of chromosomes, basic unit of compaction – Histone – scaffolding protein, building block of nucleosome – Core DNA – wrapped 1.65 times around core, ~147 bp (constant among eukaryotes) – Linker DNA – 20-60 bp (varies among eukaryotes) • DNA compaction at the nucleosome level is approximately 6-fold (far short of the 1000-10,000-fold compaction needed) • Not all DNA packaged, some is expressing, replicating, or recombining • Non-histone – transcription, replication, repair, recombination, topology VII.
    73. 73. Extract DNA from proteins Ladder in multiples of nuclease length Box 7-1
    74. 74. Histones • H2A, H2B, H3, H4 (2 each = 8 total) – Core histones – Positively-charged amino acids (K and R) make up 20% of protein – 11-15 kDa • H1 – linker histone, one copy
    75. 75. Histones • Histone-fold domain (three -helices) – A conserved region in every core histone – Mediates assembly of histones in absence of DNA Modifications Structural
    76. 76. Nucleosome Assembly • Histone assembly in the presence of DNA – H3-H4 tetramer followed by two H2A-H2B dimers – H3-H4 (big) bends DNA for H2A-H2B (small) • Site of contact between histones and DNA – H-bonds with backbone and bases of minor groove – Not sequence-specific
    77. 77. Nucleosome Assembly Time (cccDNA)
    78. 78. Negative Superhelicity • Each nucleosome adds a -1.2 change in linking number (not -1.65 because histones change number of bases per turn from 10.5 to 10.2 bp/turn • Topoisomerase relaxes remainder of DNA • Nucleosome essentially stores energy since negative supercoiling favors unwinding • Nucleosome removal – Initiation of replication – Transcription – Recombination • Prokaryotes use gyrase to induce negative supercoils (requires ATP) • Hyperthermophiles use reverse gyrase to induce positive supercoils (requires ATP)
    79. 79. Box 7-2 Topoisomerase’s role in nucleosome assembly
    80. 80. Histone-DNA Interactions H3-H4 H2A-H2B H3-H4 bind middle and both ends of DNA (essential)
    81. 81. Minor Groove Interactions • Non-specific interactions – 14 total contacts, one for each minor groove exposure – 40 hydrogen bonds between proteins and phosphodiester backbone (2X a normal DNA-binding protein) – 7 hydrogen bonds with bases in minor groove, none with any distinguishing base pairs • Basic amino acids allow phosphodiester bending (generally unfavorable)
    82. 82. Minor Groove Interactions
    83. 83. Histone Tails • N-termini • Exit nucleosome core at 11, 1, 4, 8 o’clock • Emerge between or on either side of DNA • Form grooves of a screw, directing DNA to wrap around the histone octamer in a left-handed manner • Induces negative supercoils in DNA • Recent data suggests they inhibit RNA pol II Protease treatment shows tails are accessible
    84. 84. Histone Tails
    85. 85. Histone Variants • Histones are some of the most conserved proteins • Variants change structure and function – H2A.X – widely distributed, phosphorylated upon double-stranded breaks, recognized by repair enzymes – H2A.Z – creates accessible region of the chromatin for transcription – MacroH2A – 3X larger than H2A with a leucine zipper region. Silencing? – CENP-A – replaces H3 in the centromere, binding sites for other kinetochore proteins (aurora)
    86. 86. Chromatin Structure • Euchromatin – Poor staining – Open, unorganized – High transcription – 30 nm fiber, 10 nm as RNA pol passes • Heterochromatin – Good staining – Condensed, very organized (via nucleosomes) – Low transcription – Still important – Telomere, centromere VIII.
    87. 87. Higher-Order Chromatin Structure • Histone H1 binds to linker and middle of core DNA • H1 binding results in asymmetry of DNA and core structure, the final outcome is that nucleosomes alternate on either side of linker DNA • Produces a zigzag -H1 +H1
    88. 88. Higher-Order Chromatin Structure • H1 binding to nucleosome arrays forms the 30- nm fiber – This structure is less accessible to DNA-binding enzymes (RNA polymerase, transcription factors, etc.) – Models: • Solenoid model – A superhelix with six nucleosomes per turn – A helical pitch of 11 nm • Zigzag model – Linker DNA passes through center of the fiber – Both have supporting evidence, but solenoid likely in most species
    89. 89. urnal/v13/n7/pdf/nrm3382.pd A heteromorphic fiber with predominant two-start (zigzag) type interspersed with one-start conformations was energetically more favorable than uniform zigzag or solenoid conformations under conditions that promoted the most compact folding (that is, the presence of linker histone and Mg2+ counter ions).
    90. 90. Robinson et. al., 2006. PNAS.Robinson et. al., 2006. PNAS.Robinson et. al., 2006. PNAS. Solenoid (one-start) Zigzag (two-start)
    91. 91. Robinson et. al., 2006. PNAS. “We find that over the range of nucleosome repeat lengths analyzed, there are two discrete classes of fiber structure, one 33 nm in diameter and with ~11 nucleosomes per 11 nm, and the other 44 nm in diameter and with ~15 nucleosomes per 11 nm.” Data mostly supports one-start (solenoid) model, but linker DNA determines final structure. Nucleosome More Compact Than Previously Thought Solenoid Zigzag
    92. 92. Structure Based on Linker Length
    93. 93. Higher-Order Chromatin Structure • Histone N-termini required for 30-nm fiber by interacting with adjacent nucleosomes (seen in crystal structure) • The 30-nm fiber is a 40-fold compaction of DNA • Nuclear Scaffold – Further folding of 30-nm fiber – Loops of 40-90 kbp held at the base – Base is made of non-histone proteins (mostly unknown, and even debated as an artifact) • Topo II – about 50 kb apart, at base of loop for control and topological isolation • SMC’s
    94. 94. Active transcriptionCondensed 30 nm opens into 10 nm when passed by RNA polymerase
    95. 95. Protein Scaffolding Histones/nucleosomes Topo II ATPases (remodeling) Linker proteins Others
    96. 96. Peano Curve (fractal) Overall folding affects expression, splicing
    97. 97. Regulation of Chromatin Structure • Histone association with DNA is dynamic to allow protein access • DNA unwraps rather than just coming off and reattaching • Nucleosome-remodeling complexes – Use ATP hydrolysis to slide DNA – Can also transfer nucleosome to another helix IX.
    98. 98. Nucleosome Positioning • Some are found in specific positions • Directed by DNA-binding proteins, which preferentially assemble nucleosomes nearby • If two proteins bound within 150 bp, nucleosomes cannot form (require 147 bp) • Nucleosomes are also attracted to bent DNA (A:T) • At least 50% of nucleosomes “positioned”
    99. 99. Histone Tails • Histone N-terminal and C-terminal (H1) tails may be modified (acetylases, methylases, ATPases) – Phosphorylation, acetylation, methylation, sumoylation, or ubiquitination on Ser, Thr, Lys or Arg – Affects chromatin structure and function (e.g., gene expression) – Acetylation – loosening – Methylation – silencing/repression (occasional activation) – Ubiquitination • “Histone Code” – proteins can read modifications, modify gene expression – Example: acetylation of lysines 8 and 16 of H4  expression
    100. 100. Bromo/Chromo/TUDOR/PHD/SANT domain recruitment
    101. 101. Can be cooperative or opposing
    102. 102. Bromodomain – acetylated lysines Chromodomain – methylated histones TUDOR domain – methylated histones PHD domain – methylated lysines SANT domain – histone remodeling
    103. 103. Other interaction sites:
    104. 104. Chromatin Remodeling
    105. 105. Nucleosome Assembly After Replication • Nucleosome rapidly reassembled after replication • H3-H4 tetramer, two H2A-H2B dimers, H1 • Nucleosomes must double during each chromosome duplication • Are old chromosomes all lost? If so, how would modification memory carry over to the next cell? If not, how do they separate evenly and retain this memory? – Old and new on each daughter chromosome – H3-H4 tetramers and H2A-H2B dimers either all old or all new – H3-H4 remains bound – H2A-H2B dimers released into the pool, available for new assembly – Old nucleosomes recruit enzymes that add similar modifications to adjacent nucleosomes, thus maintaining states of modification after DNA replication – Critical role in inheritance
    106. 106. Nucleosome Assembly • Histone assembly requires chaperones (not “folding” but “directing” chaperones), recognizing replicating DNA – Ex.: Rtt106 binds to H3 if acetylated at aa 56 and places H3-H4 on newly-replicated DNA • CAF-I interacts with the sliding clamp (PCNA), which holds DNA polymerase in place during replication • After polymerase finishes, the sliding clamp is released and interacts with CAF- I, which directs nucleosome assembly
    107. 107. New Evidence of a Pre-Nucleosome • ATP-dependent motor protein assembles/activates nucleosomes • Intermediate between naked DNA and mature chromatin?
    108. 108. Animation – Nucleosome Assembly New histones synthesized in S phase
    109. 109. TrxG and PcG Proteins but Not Methylated Histones Remain Associated with DNA through Replication • Study shows histones are completely removed in Drosophila • Trithorax and Enhancer-of-Zeste, which are H3K4 and H3K27 methylases, and Polycomb continuously associate with their response elements on the newly replicated DNA. We suggest that histone modification enzymes may re-establish the histone code on newly assembled unmethylated histones and thus may act as epigenetic marks.
    110. 110. Epigenetics • Changes in gene expression (not sequence) based on environment via histone modification (imprinting, silencing, inactivation). Most removed in zygote, but some can last generations for fast phenotypic adaptation • ~100 genes known, relevant in development, cancer, disease, stem cells, exercise, drug abuse • Histone modifications play a role in autism, schizophrenia, depression, and other psychiatric diseases, and the H3K4-specific histone demethylase, JARID1C/SMCX, has been linked to mental retardation and autism • Disease – 15q11 maternal/paternal imprinting, variations cause Prader-Willi or Angelman Syndrome • Starving Dutch mothers who gave birth during WWII famine had children who were more susceptible to obesity and other metabolic disorders – and so were their grandchildren • Swedish grandsons (but not granddaughters) had lower cardiovascular disease if their grandfather had gone through famine • Rats – chronic high-fat diets in fathers result in obesity in their female offspring; obese diabetic mice altered pancreatic/fat gene expression in offspring • Mice – paternal stress at any point in their lives marks sperm via microRNA, causing hypothalamic–pituitary–adrenal (HPA) axis dysregulation in offspring • Lamarckism? Bees and methylation Epigenetics: http://www.youtu =LcaRTDsLmiA
    111. 111. Toxins and Epigenetic Changes via DNA Methylation • Exposed pregnant mouse to toxins, studied F1-F3 • Early-onset puberty, egg/sperm reduction in F3 (promoter methylation) • Your great-grandparents’ exposure could be causing diseases epigenetically Hundreds of genes affected Dioxin (red) Pesticide (light blue) Plastic (pink) Jet fuel (dark blue)
    112. 112. Influenza Controls Gene Expression via Histone Mimicking • Immunosuppressive NS1 protein of the influenza A virus mimics a core component of gene regulating machinery in order to block antiviral gene function • NS1 protein of the H3N2 strain of influenza -- the "seasonal" flu -- contains the same sequence of amino acids as the "tail" domain of a DNA packaging protein in humans called histone H3. The histones are present in the cell nucleus and play an important role in gene activation. Chemical modifications of the histone "tails" allow recruitment of effector proteins that, in turn, determine which genes are switched on or off • Impairs host antiviral response