BIMM 100 Lecture 12

BIMM 100 Lecture 12 - Lecture 12: Looking deeper;...

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Unformatted text preview: Lecture 12: Looking deeper; chromosomes and chroma7n Reading: pages 247 ­257 Using what you’ve learned… Possible other applica7ons? HIV! Development of flu vaccine now at UPENN ­ 30,000 people a year die from influenza, and an effec7ve vaccine has never been produced! DNA vaccine: 1.  Made a plasmid library of influenza an7gens (because they are so diverse) 2.  Inject the plasmids into pa7ents 3.  Plasmids will be targeted to the immune system, where the immune cells will produce an7bodies against them DNA packaging The “problem” with DNA: a single human cell can contain 2 meters of DNA! All of this must be contained in a cell that is, on average, only 10 microns in size! How is this done? Chroma'n! DNA is packaged in an organized fashion ­ it needs to be readily available for replica7on, repair, and transcrip7on. DNA organiza7on in eukaryo7c cells We’ll be discussing: Histones & Nucleosomes (beads on a string) histone modifica7ons different types of chroma7n (euchroma7n vs. heterochroma7n) Loops of the 30nm fibers Higher order chroma7n structure Chromosome packaging: the animated version Chroma7n: what is it? Simple answer? Comprised of ~50% DNA, and ~50% protein Beads on a string Low salt condi7ons 30nm fiber Physiological condi7ons Nucleosomes: composed of DNA and histones ­ primary structural units of chroma7n •  10nm in diameter •  DNA is wound 1 2/3 7mes around the histone core •  147 bps per nucleosome •  Linkers vary in length (10 ­90 bps) Histones Majority of protein in chroma7n Small (but abundant!) proteins Contain lots of posi7vely charged amino acids Core histones are highly conserved among species •  Gene families •  •  •  •  –  Mul7ple copies in the genome Histones and the nucleosomes Histones: 5 major types Core: an octamer containing two copies each of H2A, H2B, H3, and H4 Linker: H1 H2A/H2B dimer + H3/H4 dimer = Octameric histone core + DNA and H1 = Nucleosome! Structure of the nucleosome: DNA wraps around it DNA “covers” most of the protein on the nucleosome’s lateral surface. Histone tails are not shown here… Chroma7n can be extended or compacted Extended: chain of nucleosomes •  Usually associated with “ac7ve” DNA (being transcribed or replicated) Compacted: 30nm fiber (includes the H1 histone) •  Usually associated with “inac7ve” DNA Lem handed helix! Chroma7n structure •  Very well conserved ­ similar in: –  Fungi –  Plants –  Animals •  Likely essen7al for the development of eukaryo7c cells –  Significant devia7ons were likely strongly selected against during evolu7on –  Core proteins have similar 3D conforma7ons •  One major difference? –  H1 varies more from organism to organism Epigene7cs •  Process that affects the expression of specific genes (and is inherited by daughter cells) that does not result from changes in the DNA sequence •  Example: Historical study in Sweden showed that male children that enjoyed overabundant winters (i.e. kids who went from normal ea7ng to glupony in a single season) produced sons and grandsons who lived shorter lives. How could this be? –  If this were gene7c, it should take genera7ons to occur, right? Great ar7cle: “why your DNA is not your des7ny” Remember: epigene7cs is not evolu7on! It is not passed down to new genera7ons. It appears to just be a rapid altera7on of gene expression in response to a s7muli. Known about since the 1970’s ­ no conclusive experiments performed un7l 2003! Scien7sts at Salk claim to have complied a “map of the human epigenome” hpp://www.7me.com/7me/magazine/ ar7cle/0,9171,1952313,00.html Clinical importance? •  Azaci7dine, a nucleoside analog of C, are used in the treatment of myelodysplas7c system (MDS). •  How does it work? –  acts as a false substrate and potent inhibitor of methyltransferases leading to reduc7on of DNA methyla7on Histone tail modifica7ons: a way that epigene7cs could be occurring •  Pospransla7onal modifica7ons •  Responsible for chroma7n condensa7on (or decondensa7on) •  Tails are responsible for chroma7n condensa7on Types of modifica7ons? Methyla7on (R, K) Acetyla7on (K) Ubiqui7na7on (K) Phosphoryla7on (S, T) Performed by enzymes: histone methyl transferases (HMTs), histone acetylases (HATs), histone deacetylases (HDACs) The histone code hypothesis •  Different modifica7ons (or combina7ons of modifica7ons) create specific binding sites for proteins Protein Y Ac Protein X Me K16 H3 K16 H4 The recruitment of different proteins may have an effect on gene expression! Heterochroma7n and euchroma7n •  Heterochroma7n –  Tightly condensed –  Less accessible to proteins –  Transcrip7onally inac7ve •  Euchroma7n –  Open chroma7n –  More accessible to proteins –  Transcrip7onally ac7ve Heterochroma7n and euchroma7n •  Heterochroma7n –  Tightly condensed –  Less accessible to proteins –  Transcrip7onally inac7ve •  Euchroma7n –  Open chroma7n –  More accessible to proteins –  Transcrip7onally ac7ve Heterochroma7n and euchroma7n: different modifica7ons •  Lysine’s ε ­amino group can be acetylated, neutralizing it’s posi7ve charge •  Conversely, lysine’s ε ­amino group can also be methylated, which prevents acetyla7on, maintaining their posi7ve charge Histone acetyla7on at lysine residues CH3 CO NH3+ NH CH2 CH2 CH2 CH2 HATs CH2 CH2 +H 3N CH2 CH2 C COO ­ Lysine H +H 3N C H COO ­ Acetylated lysine •  Acetyla7on occurs at the e ­amino group •  Performed by HATs •  add acetyl group from acetyl ­CoA •  Neutralizes + charge on lysine •  Recognized by bromo ­ domain proteins •  Important for chroma7n decondensa7on and euchroma7n forma7on Euchroma7n and histone acetyla7on Specific chroma7n modifica7ons signal to make chroma7n euchroma7c H4K16 acetyla7on Allows transcrip7onal ac7vators to bind (like TFIID) Histone methyla7on at lysine residues CH3 NH2 NH3+ NH CH2 CH2 CH2 CH2 HMTs CH2 CH2 +H 3N CH2 CH2 C COO ­ Lysine H +H 3N C H COO ­ Methylated lysine •  Methyla7on (mono ­, di ­, or tri ­) occurs at lysines and arginines •  HMTs add methyl groups •  Maintains + charge on lysine •  Recognized by chromo domain proteins •  Important for chroma7n condensa7on and heterochroma7n forma7on Forma7on of heterochroma7n •  There is a specific signal for heterochroma7n forma7on: –  H3K9 methyla7on •  HP1 (has a chromo domain) binds to this methyl group Heterochroma7n forma7on is propagated along a chromosome This should shut down all chroma7n ­ but, it doesn’t! Why? Boundary elements provide barriers! Usually they are non ­ histone proteins bound to specific genomic regions that limit the spreading of both heterochroma7n and euchroma7n Important experimental technique: DNAse I treatment How can euchroma7n and heterochroma7n differences be observed experimentally? –  DNAse I randomly digests DNA –  DNA in euchroma7n is sensi%ve to DNAse I treatment –  DNA in heterochroma7n is resistant to DNAse I treatment DNAse I diges7on: the principle Principle? •  That DNA that is euchroma7c (ac7ve) will be more decondensed (or “open,” and thus more susep7ble to DNAse I diges7on DNAse I diges7on: the experiment Probe size 1.  Isolate nuclei of cells (to get chroma7n) 2.  Treat DNA with DNAse I 3.  Isolate DNA (separate from protein) 4.  Perform Southern Blot! Probe can’t even anneal to anything ­ it is all destroyed! Higher order chroma7n structure: the 30nm fiber •  Nucleosomes are further compacted into the 30nm fiber •  How? –  chromosome scaffolds comprised of nonhistone proteins –  DNA is arranged in large loops that apach to a protein scaffold at scaffold associated regions (SARs) or matrix apachment region (MARs), which are located between transcrip7onal units Chroma7n loops Electron microscopy Done by FISH! Each dot represents a probe ­ Dot that were physically far apart appeared close to one another A B C D E F G H Protein scaffold is comprised of SMC proteins ATP ac7vity Structural maintenance of chromosome proteins (SMCs) •  nonhistone proteins important for chroma7n structure •  form rings around the chroma7n fibers Chroma7n changes during the cell cycle Interphase: Chroma7n is decondensed Clear chromosomal territories (chromosomes don’t stretch out throughout the whole nucleus) Metaphase: Chroma7n is condensed Ready for cell division Model describing the folding of the chroma7n fiber that occurs before metaphase (during prophase) •  Even more compac7on occurs! •  Cri7cal for packing! X ­chromosome inac7va7on Reminder: –  Males XY –  Females XX •  Great example of heterochroma7n forma7on that correlates with gene inac7va7on: DNA becomes condensed and inac/ve –  Females inac7vate one of their X chromosomes •  Random –  Occurs during embryonic development –  Maintained through mitosis/DNA replica7on –  mosaic X ­chromosome inac7va7on Xa (ac7ve), and Xi (inac7ve) •  Epigene7c ­ not coded in the DNA •  Uses noncoding RNA Xist, which coats the X chromosome that it was coded from, silencing it. •  Inac7ve X chromosome is called a Barr body. •  Not fully understood/ characterized! Chromosome characteris7cs •  Chromosomes are duplicated sister chroma7ds •  Karyotype –  number, size, and shapes of the chromosomes –  Observed during metaphase –  Species ­specific •  Autosomal vs. sex chromosomes •  Telomeres and centromeres How can we iden7fy individual chromosomes? •  Chromosome “pain7ng” –  Use specific probes for genes on chromos –  Modify using a unique fluorescent output for each probe Dye banding paperns Giemsa (stains DNA) stains “G bands” Low G ­C content DNA stains more intensely Chromosome pain7ng and banding are essen7al for understanding the evolu7on of chromosomes •  The experiment? TREE SHREW –  Took FISH probes directed towards genes on the tree shrew’s 16th chromosome –  Applied these to a human metaphase spread to see where they would anneal •  The result? HUMAN –  The tree shrew’s 16th chromosome has the same genes that we have on our 10th chromosome! Chromosome pain7ng and banding are essen7al for understanding the evolu7on of chromosomes •  The importance? TREE SHREW HUMAN –  These results indicate that somewhere during the evolu7on of humans and tree shrews (about 85 million years), a long, con7nuous DNA sequence on an ancestral chromosome became chromo 16 (in tree shrews) and chromo 10 (in humans). The term for this? Synteny! •  Synteny –  La7n for “on the same ribbon” –  Presence of two or more genes in a common chromosomal region in two or more species indica7ng a common chromosomal segment Actual experiment: Gcsf (again!) This 7me it’s labeled as CSF3 (for it’s original name ­colony s7mula7ng factor 3) CSF3 synteny conserved among humans, mice, opossums, chicken, finches, and lizards. Looks like they’re two copies in fish (a and b) that evolved millions of years before these other organisms! Bartunek Can use these techniques to determine the evolu7on of chromosomal regions •  No significant rearrangements (H1) •  Fusion (H2 from P9 & 11) •  Breakage (H14 & 15 from P5) •  Transloca7ons (H12 & 22 from P14 & 21) DNA organiza7on in eukaryo7c cells Almost done Chapter 6: Star7ng Chapter 7 soon! •  Histones and nucleosomes (beads on a string) –  Histone mods –  Heterochroma7n vs. euchroma7n •  Loops of 30nm fibers •  Higher ­order chroma7n structure For your enjoyment: the Histone song… For when you’re bored and studying for the final! Actually published as an “ar7cle” in the journal Developmental Biology ...
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