Presentation on theme: "Eukaryotic chromosomes"— Presentation transcript:
1Eukaryotic chromosomes Bacterial EukaryoticDNA is in a nucleoid body DNA is in chromosomesThere is one large DNA molecule There are many moleculesCircular LinearThe DNA in the diploid nucleus is ~2 meters long.It is present in a nucleus that is a 1000 cubic microns.Function of chromosomesPackagingRegulationTotal human DNA is 3x109 bpSmallest human chromosome is 5x107 bpThe DNA in this chromosome is 14 mm longThe chromosome is 2um long7000 fold packaging!
2Amount of DNA varies between species Amount of DNA varies in eukaryotesSalamander genomes are 20 times larger than human genomesBarley genome is 10 times larger than the rice genomeBarley and rice are related.Measurements of DNA lengthAmount of DNA/nucleus = C valueSpecies DNA content (pg or 10-12g)haploidSpongeDrosophila 0.2Human 3.5Lungfish 102Locust 46Frog 4.2Yeast
3C-Value paradox This is often called the C value paradox. There is no phylogenetic relationship to DNA contentThere are sibling amphibian species - they look morphologically identical but have 4-fold difference in DNA contentHow do we account for the differences in DNA content/nucleusNo of genesGene sizeDistance between genes
4Junk DNA 1) Number of genes could vary in these organisms Lungfish would have to have 30 fold more genes than humansBarley and rice have the same number of genes but vastlydifferent DNA contents.Number of genes does not correlate with amount of DNA in a cell.2) Size of genes could increase as genomes increaseDrosophila genome is 30 times larger than E.coliAverage coding region of a gene is 1-2 kb long in DrosophilaE. Coli genes are only slightly shorterDrosophila genes are not 30 times larger than E. coli genes.Introns and promoters etc increase the size to some extent but cannot account for all of the increase.3) Amount of DNA between genes increasesHumans= 25,000 genes. Size of human genome is 3x109 bpYeast= 6000 genes. Size of yeast genome is 1.4x107 bpThe DNA between genes (intergenic region) varies.A large fraction of intergenic DNA is repetitiveNearly 60% of the human genome is repetitive.Less than 5% of the yeast genome is repetitive.
5Genome Human Gene catalog Vertebrates 46% Eukaryote & Prokaryote 21% Human specific<1%Eukaryotes32%Human Genes are categorized according to their function, as deduced from the protein domains specified by each gene.
6Human Genes Categorization of human genes Genes whose function is not known are omitted
8Differentiation Epigenetics and development n + n 2n DNA content same DNA content,> 200 cell types
9Gene packaging and why is it important Epigenetics: Gene regulation through stable repressionWolffe and Matzke, Science, 1999
10Chromatin A human cell contains about 2 m of DNA (1 m per haploid) The human body consists of approximately 1013 cells and therefore contains a total of about 2 × 1013 m of DNA.Distance from the earth to the sun is 1.5 × 1011 mThe diameter of the nucleus is 5x10-6 metersHow is the DNA packaged?Chromatin= DNA +histones +non-histones1g 1g 1g
12Nucleosomes Four histone proteins H2A H2B H3 H4 Very highly conserved There are two copies of each core histoneDNA is wrapped around the outside of the histone octamer166 bp of DNA wraps around the histonesLinker DNA connects nucleosomes1 mol of linker Histone H12 mol H2A2 mol H2B2 mol H32 mol H41 mol H1~200 bp DNAA eukaryotic chromosome made out of self-assembling 70A unit, 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!(Anonymous review of Woodcock Ms 1973, published in 1975)
15Differentiation Epigenetics and development n + n 2n DNA content same DNA content,> 200 cell types
16All Genes are poised for activity spermeggEmbryoAll Genes are poised for activityCell commitmentSpecific genes activatedAll other genes inactivatedActive genes maintain activityInactive genes remain silentActive genes maintain activityInactive genes remain silent
17Transcription activator +++ --- Repressor proteins --- +++ Epigenetics and epigenetic regulationHeritable changes in gene expression that do not involve changes in DNA sequencesGenes contain coding regions and promoters. examples:Developmentally regulated / tissue specific gene expressionX chromosome dosage compensationGene ImprintingPosition effect variegation (PEV). mechanisms:Changes in Transcription factorsChanges in DNA methylationChanges in ChromatinActive InactiveTranscription activatorRepressor proteinsDNA methylationHistonesHistone Acetylation
18examples:Developmentally regulated / tissue specific gene expressionX chromosome dosage compensationGene ImprintingPosition effect variegation (PEV)Gene activationCell/tissue specific transcriptional activators bind to enhancers of genes that have binding sites for these factors-Aid in recruitment of enzymes that modify chromatin at the promoter- Aid in recruitment of the general transcription machinery and RNA polymeraseInrTATAGenePromoterEnhancerThe enhancer functions to activate genes. There are specific sequences that bind TISSUE SPECIFIC transcription factors. The binding of these factors induces gene activation 100 fold!
19Cell specific expression Different Enhancers bind different tissue and cell specific transcription activator proteins and this enables specific gene activation in specific cellsHNF3Liver CellLiver gene1Liver gene2Brain gene1Brain gene2Brain CellNZF2Liver gene1Liver gene2Brain gene1Brain gene2
20All Genes are poised for activity spermeggEmbryoAll Genes are poised for activityCell commitmentSpecific genes activatedAll other genes inactivatedActive genes maintain activityInactive genes remain silentActive genes maintain activityInactive genes remain silentmechanisms:Changes in Transcription factorsChanges in DNA methylationChanges in ChromatinDNA methylationRepressor proteinsHistones
21De-differentiation Differentiation Epigenetics and development 2n DNA contentDe-differentiationsame DNA content,> 200 cell typesDifferentiationCloning by nuclear transfer --> regenerate entire organism from transfer of single nucleus (e.g. Dolly)Induced pluripotent stem cells (iPS) --> expression of 4 genes are sufficient to transform differentiated cells to “stem” cellsBoth processes must involve reprogramming of epigenome!
22Inactive chromatinHeterochromatinInactiveEuchromatinActiveConstitutive heterochromatin: Repetitive DNA-Centromeres, telomeres etcRepetitive DNA tends to recombine expanding/contracting repeats. Preventing repetitive DNA from recombination is critical for cell survivalConstitutes ~ 20 % of nuclear DNAHighly compacted,Always transcriptionally/Recombinationally inertEuchromatin + facultative heterochromatin:constitutes ~ 80% of nuclear DNAless condensed, rich in genes,Euchromatin is transcriptionally activethe rest is transcriptionally inactive (but can be activated in certain tissues or developmental stages)These inactive regions are known as “facultative heterochromatin”
23Gene Silencing and its importance In any given cell, only a small percentage of all genes are expressedVast majority of the genome has to be shut down or silencedKnowing which genes to keep on and which ones to silence is critical for a cell to survive and proliferate normally during development and differentiationTranscription factors bind active genes and keep them activeDNA methylation of inactive genes keeps them inactiveCell commitmentSpecific genes activatedAll other genes inactivatedActive genes maintain activityInactive genes remain silent
24DNA Methylation is not perfectly inherited during development/aging Twins Monozygous twins share a common genotype. most monozygotic twin pairs are not identical, differences in susceptibilities to disease and a wide range of anthropomorphic features. Examined the global and locus-specific differences in DNA methylation and histone acetylation. Twins are epigenetically indistinguishable during the early years of life, but older monozygous twins exhibited remarkable differences.The non-methylated sites were cut in an initial digestion using the methylation-sensitive SmaI, which leaves blunt ends. (SmaI is a methylation-sensitive enzyme that does not cleave DNA when central cytosine in –CCCGGG- is methylated).A second digestion was performed using the isoschizomer PspAI, (PspA1 is not sensitive to methylation) which leaves a CCGG overhang. Adaptors are ligated to the sticky ends. DNA fragments flanked by two ligated adaptors were amplified by PCR using specific primers that hybridize to the adaptor.To study the distribution of the AIMS amplified DNA along the chromosomes, we hybridized equimolecular quantities of each twin AIMS product. One twins DNA was labeled with spectrum red, the others with spectrum greenFraga et al., 2005PNAS 102(30):
25Facultative heterochromatin Regions of genome, rich in genes that are condensed in specific cell types or during specific stages of developmentIt includes genes that are highly active at a particular stage of development but then are stably repressed.X-chromosome inactivation in vertebratesDosage compensationNo. of transcripts are proportional to no. of gene copiesDiploid- 2 copies of a geneGenes on X-chromosomesIn females there are two copies of a gene. In males there is one copy.XX XY2 1Measuring transcript levels for genes on the X chromosome in female and male show that they are equivalent.Dosage imbalance is corrected!
26examples:Developmentally regulated / tissue specific gene expressionX chromosome dosage compensationGene ImprintingPosition effect variegation (PEV)Dosage compensationIn Drosophila in the males there is an increase in transcription from the single X chromosome. A inhibitor of transcription is turned off in males allowing for full expression from the one X chromosomeIn nematodes there is a decrease in transcription from bothX chromosomes- protein binds the 2X chromo and causes chromosome condensation which reduces transcription.In mammals, X chromosome inactivation occurs in females by formation of heterochromatin on one X chromosome
27Mammalian X-chromosome inactivation Mammalian males and females have one and two X chromosomes respectively.One would expect that X-linked genes should produce twice as much gene product in females compared to males. Yet when one measures gene product from X-linked genes in males and females they are equivalent.This phenomenon, known as dosage compensation,X chromosome inactivation in females is the mechanism behind dosage compensation.In females, one of the X chromosomes in each cell is inactivated. This is observed cytologically. One of the X-chromosomes in females appears highly condensed. This inactivated chromosome is packaged into heterochromatin and forms a structure called a Barr-body.
28Dosage compensationDosage compensation in mammalian females occurs by shutting off of most of the genes on one X chromosome in females.The inactive X chromosome becomes heterochromatic.It is called a Barr bodyXCI is random.It occurs at the 500 cell stage of the embryoFor a given cell in a developing organism, probability of the maternally or paternally derived X being inactivated is equal.Once inactivated, it is stably propagated so that all the thousands or millions of cells descended from that embryonic cell maintain the same chromosome in the Heterochromatic state.Xist is ON - Xist RNA coats the X- X chr is OFFTsix is on- Tsix pairs and inactivates Xist -X chr is ONX chr with Xist gets methylated!!!!!
29XYXXreactivateXeggXspermXXXXXXTsix ActiveXist ActiveXist RNAInactivatesXist RNACoat inactive X- methylate DNA
30Epigenetic mechanism #1: DNA methylation DNA methylation has long been correlated with repression of gene expressionDNA methylation mostly occurs on CpG dinucleotidesDNMTsmethyl group added to the cytosinemethylation status is maintainedduring replication/mitosis
31X-inactivationThe inactivation of one of the two X-chromosomes means that males and females each have one active X chromosome per cell.X-chromosome inactivation is random. For a given cell in the developing organism there is an equal probability of the female or the male derived X chromosome being inactivated.
32X-inactivation zygote Inactivation Embryo The embryo is a mosaic! Once the decision is made in early development, then it is stably inherited.Patches of cells have the male X ON and patches of cells have the female X ONThis is a Developmental rule that overlays on top of Mendellian rules!
33Barr bodies· The inactive X-chromosome in normal females is called the barr body. XXX individuals have 2 Barr Bodies leaving one active X· XXXX individuals have 3 Barr Bodies leaving one active X· XXY individual have one Barr Body leaving one active X(Klinefelter's syndrome)· X0 individuals have no Barr Bodies leaving one active X(Turner's syndrome)Given X-chromosome inactivation functions normally why are they phenotypically abnormal?Part of the explanation for the abnormal phenotypes is that the entire X is not inactivated during Barr-Body formation (Escape loci)Consequently an X0 individual is not genetically equivalent to an XX individual.XX femaleXXX femaleXXY maleXY male
35Tortoise shell cats Orange Black Enzyme O The O gene is carried on the X chromosome.Female cats heterozygous for the O gene on the X- chromosome have a particular pattern called Tortoise shell.According to Mendel’s rules the cats should be either orange or black.But the cats are neither! They are Tortoise shell.
36Tortoiseshell catsAll tortoiseshell cats are femaleXY maleIf normal O gene is present on the X, the male is gingerotherwise he is blackFemale with O/O are gingerFemales with o/o are blackFemales with O/o are tortoiseshellIn O/o femalesX-chromosome inactivation happens at randomSome cells activate O gene making ginger pigmentSome cells activate o gene making black pigment
37Tortoise shell catsAccording to Mendel’s rules these cats should be either orange or black. But the cats are neither! They are Tortoise shell.OO x oYF1 females are Oo
38Tortoise shell catsFemale cats heterozygous for the O gene on the X- chromosome have a particular pattern called Tortoise shell. According to Mendel’s rules these cats should be either orange or black. But the cats are neither! They are Tortoise shell.
39Imprinting Occurs on Autosomes Occurs only on some genes on autosomes examples:Developmentally regulated / tissue specific gene expressionX chromosome dosage compensationGene ImprintingPosition effect variegation (PEV)ImprintingOccurs on AutosomesOccurs only on some genes on autosomes
40Calliphyge-eautiful Oklahoma 1983Farmer observed sheep with Calliphyge!!!Beautiful Behinds!!He thinks---Ooh la la--*!*!*! $$40% more muscle7% less fat20% increased profit
41CalliphygeeautifulBig bottom male X normal female203 big bottom:209 normalCalliphyge is Sex independent(both males and females are big bottom)Big bottom is autosomal dominant?
42Big bottom male x normal female 203 big bottom:209 normalCNC : NN N50%NC100% normalNot Sex linked- The callipyge gene is on autosome
43CC x NN 100% Callipyge NN x CC 0% Callipyge Calliphyge gene is expressed when inherited from the males!!!The calliphyge locus from mother is always silenced.
44Callipyge The callipyge locus from mother is always silenced. Normal female X Normal maleNormal phenotypefemale allele is imprinted (turned off) and male allele is expressedThe callipyge locus from mother is always silenced.Normal female X mutant malemutant female X Normal male*Normal phenotypeMutant allele (from mom) is imprinted (turned off) and normal allele (from dad) is expressed*Mutant phenotypeNormal allele (from mom) is imprinted (turned off) and mutant allele (from dad) is expressed
45A small number of genes (~200) on autosomes ImprintingA small number of genes (~200) on autosomesThe allele from one parent is shut off.In the egg/sperm, these genes are imprinted (turned off)Imprinting leads to functional haploidy!Gene is WT but no protein is made (i.e. mutant).Abandoned safety net of diploidy.GameteA=offA=offA=onSomatic cellA=onThe original imprint is erased in gametes and the new imprint is established in progeny during gamete formation
47Imprinting of the IGF-2 gene on chromosome 11p15. Boundary model of the IGF-2 and H19 cluster on chromosome 11p15. IGF-2 and H19 share a common enhancer downstream of H19. On the maternal allele, the ICR upstream of H19 is unmethylated and binds the vertebrate enhancer-blocking protein CTCF, which inhibits the activation of the IGF-2 promoter by the enhancer. On the paternal allele, the H19 promoter and ICR are methylated, thus silencing H19 and interfering with CTCF binding. The IGF-2 promoter is activated by its enhancer (30 , 31) .Schneider D T et al. Cancer Res 2001;61:
48War of the sexes Why are perfectly good genes turned off? Many maternally imprinted genes (inactive on the maternal chromosome) are fetal growth factor genesTug of warFather contributes active genes to enhance growth- extract as many maternal resources for offspring as possible. He is unlikely to mate again with that female. Advantage for survival of his gene pool.Mother silences these growth promoting genes to ration her investment to any one offspring conserving resources for future.
49DLK1 GTL2 PEG11 MEG8 effector repressor M Mc M Mc P P Pc Pc Callipyge locusDLK1GTL2PEG11MEG8effectorrepressorMMcMMcPPPcPc
50ModelSusan K. Murphy et al. Genome Res. 2006; 16:
51Don’tCN x CNIf C is autosomal dominantCallipyge : normal3:1CC CN NC NNActual ratio isCallipyge : normal1:3CC CN NC NN
57Heterochromatin Euchromatin Heterochromatin Most heterochromatin is made up of repetitive DNA sequencesand retrotransposon repeats.Small 5-12 bp DNA sequences repeated 100,000s timesCommon sequences of the repeatsDrosophila Yeast Human(AACAA)n (TGGG)n (CAGGG)n(AATAG)n (GGGCAAAAA)n(AATAAAC)n (GGAA)n
58Heterochromatin Heterochromatin is concentrated around Centromeres- centromeric heterochromatinTelomeres- telomeric heterochromatinHeterochromatin on chromosome arms- intecalarySome chromosomes are almost entirely heterochromaticDrosophila Y chromosomesSpecific proteins bind DNA in heterochromatin.Genes in heterochromatin are inactive.Recombination is reducedIn yeast <10% of genome is heterochromaticIn Drosophila 30% of genome is heterochromaticIn humans > 50% of genome is heterochromaticWhat is the function of heterochromatinHeterochromatin is found in all eukaryotes**In some organisms loss of heterochromatin is not lethalTherefore its role must not be very important?!**Loss of heterochromatin affects longevity (shortens life span)**Loss of heterochromatin increases non-dysjunction
59Heterochromatin Since heterochromatin reduces recombination Is increases in genome size due to loss of recombinationcontrol?What is the function of junk DNA/heterochromatin?Majority of the DNA is useless????***Anti-parasitic?Retrotransposons insert into the genome, hop around and aremutagenic. Heterochromatin would prevent their insertion,hopping around and thereby reduce mutations.Buffer against mutagenesis?Plasticity during evolution?
60Facultative heterochromatin Regions of genome, rich in genes that are condensed in specific cell types or during specific stages of development It includes genes that are highly active at a particular stage of development but then are stably repressed.X-chromosome inactivation in mammals.Dosage compensationNo. of transcripts are proportional to no. of gene copiesDiploid- 2 copies of a geneXX XY2 1Measuring transcript levels for genes on the X chromosome in female and male show that they are equivalent.Dosage imbalance is corrected!In nematodes there is a decrease in transcription from bothX chromosomes- dpy27 binds the 2X chromosomes and causes chromosome condensation which reduces transcription.In Drosophila in the males there is an increase in transcription from the single X chromosome. A inhibitor of transcription is turned off in males allowing for full expression from the one X chromosomeIn mammals, X chromosome inactivation occurs in females by formation of heterochromatin.
61Mendocino 2004 Prop H-Ban use of genetically modified organisms DNA or Deoxyribonucleic acid means a complex protein that is present in every cell of an organism and is the blueprint for the organisms development