Chromosome structure and function Side notes: Top hat 1 Which of the following statements is true? • Proteins cannot interact with the DNA backbone • All proteins with an alpha helix will bind to DNA • DNA-binding proteins will only bind sites that exactly match the recognition sequence • Beta strand domains can serve as DNAbinding domains Top hat 2 Why is there so much repetitive DNA in the human genome? • Transposable elements have rapidly copied themselves across the genome during human evolution • Lack of evolutionary pressure on genome size allows nonfunctional elements to accumulate • Some of the repetitive elements may serve functional roles • All of the above *Condensed chromosomes have characteristic lengths and banding pa erns DNA is packaged into chromosomes DNA is packaged into linear or circular units called chromosomes Eukaryotes = linear chromosomes Most bacteria & archaea = circular chromosomes In eukaryotes, DNA is wrapped round packing proteins (histones) and compacted in a space-efficient way Some cells have small extra pieces of DNA The genome is the collection of all chromosomes in a cell A gene is a region that controls a discrete hereditary characteristic, usually a speaker product like protein Genome size varies widely across organisms Genomes vary widely in the size of genome, number of genes and gene spacing E. coli is 98% genic Single-celled organisms generally have fewer genes than multicellular organisms Larger genome size and increased genome complexity do not always correlate Genomes often have transposable elements - pieces of DNA that copy themselves within a genome and lead to increases in genome size Eukaryotic genome composition Yeast genome: 12 Mbp size 16 chromosomes ~5,800 genes (+ ~400 pseudogenes) Human genome: 3,000 Mbp size 23 chromosome pairs ~ 20,000 genes Cell cycle: duplication & segregation of chromosomes Chromosomes must be copied and transferred to the next generation of cells faithfully Cells must copy DNA and separate the chromosomes before they can divide The eukaryotic cell cycle has four stages G1 (gap phase 1) - cells grow to a minimum size require for the next stage S (synthesis phase) - DNA is copied, duplicating the chromosomes. The two copies (sister chromatids) remain associated with one another G2 (gap phase 2) - the cell prepares for mitosis M (mitotic phase) - sister chromatids are separated and a full set of chromosomes is moved to each pole of the cell (chromosome segregation) Chromosomes are segregated during M phase On entry to M phase, the nuclear membrane breaks down Chromosomes condense, but sister chromatids are still associated Centromeres of the sister chromatid bind to microtubules from each pole The associations between sister chromatids are removed and the sister chromatids move apart to opposite poles of the cell Chromosomes have a distinct appearance at mitosis Chromosome structure and function Side notes: A karyotype is a display of the chromosomes from an individual, derived from microscopy images prior to separation of sister chromatids How many chromosomes are in a cell? Ploidy refers to now many sets of identical chromosomes an organism has Humans are diploid - two copies of chromosomes 1-22 and either two X chromosomes or one X and one Y Human egg and sperm cells are haploid, and only have one copy of chromosomes 1-22 plus an X or Y A small number of human cells (e.g. Hepatocytes) are polyploid, and have multiple sets of each chromosome Eukaryotic cells can contain DNA outside of the nuclear chromosomes: Mitochondria, which provide cells with ATP, have their own chromosome Chloroplasts, which derive energy from sunlight in plants, have their own chromosome Mitochondria and chloroplasts are thought to have derived from ancestral free-living bacteria that were absorbed and maintained by other single-celled organisms Bacterial cells often have additional DNA within their cells: Plasmid = small circular DNA molecule. Often encode a few proteins that confer a selective advantage, such as antibiotic resistance DNA is packaged by nucleosomes in chromatin Chromosomes are made up of chromatin; a DNA-protein complex that condenses DNA In eukaryotes, the basic binding proteins are called histones There are four core histones - they arose early in eukaryotic evolution and, as they are crucial for survival, are very highly conserved Histories are rich in lysine and arginine which have positive charges and stabilize the DNA-histone interaction DNA wraps 1.67 times around a histone octamer to form a nucleosome - this is the basic repeating unit of chromatin The histone octamer has two each of the four core histones H2A, H2B, H3, and H4 The H3-H4 heterotetramer associates with DNA The two H2A-H2B dinners then associate to form the octamer The DNA is wound around the histone octamer in a left-handed direction when the octamer is removed, this leaves negatively supercoiled DNA Negative supercoiling makes strand separation easier, which is required for replication and transcription Histone variants may have distinct functions Other histone variants can be incorporated into nucleosomes, and are found at special chromatin locations For example, H2A.X is phosphors rated at sites of DNA double-strand breaks and recruits repair machinery MNase digests non-nucleosomal DNA Micrococcal nuclease is an endo-exonuclease isolated from staphylococcus aureus. It non-specifically digests unprotected DNA Endo-nuclease: cuts DNA Exo-nuclease: chews DNA from end Exposed double-stranded DNA is digested by the nuclease. Regions wrapped Side notes: Top hat 3 What does this shorthand description of a histone modification mean: H3K9me3 • Histone 3 has been modified at the ninth lysine with three methyl groups • The ninth histone protein in the H3 family has been triply methylated • Histone 3 has been modified at the ninth lysine with three acetyl groups around histories are protected from digestion After digestion, the remaining intact DNA can be visualized by gel electrophoresis MNase-seq tells us the organization of nucleosides The Pugh lab at Penn State used sequencing methods to identify the DNA sequences wrapped around histories Histones were chemically linked to DNA DNA was digested with (MNase) Histone-linked DNA was isolated using histone antibodies (anti-H3) Proteins were removed and the DNA sequenced The DNA was mapped onto chromosomes to find the locations of nucleosides Nucleosome signals were averaged across all protein-coding genes This showed that promoter regions of yeast genes are relatively free of histones It also showed that nucleosomes are well-positioned at yeast genes The same group also looked at where the H2A.Z histone variant is located, finding that it is specifically incorporated at the first nucleosome in gene bodies Histone tails can be chemically modified Each core histone has an N-terminal "tail" that extends outwards between the DNA coils Tails are up to 25 amino acids, and have an undefined structure Tails interact with other nucleosomes to help compact DNA further Histone tails can also be chemically modified - these modifications are important for chromatin structure and function Histone side chains are subject to modifications that act as signals during regulation - acetylation, methylation, phosphorylation and ubiquitination Modifications are thought to recruit specific proteins to the chromatin modifications of unstructured histone tails are common Histone modifications are reversible - specialized enzymes add and remove the chemical groups Histone modification enzymes add/remove histone marks Histone acetyltransferases (HATs) add acetyl groups to lysine side chains. Histone deacetylases (HDACs) remove acetyl groups Histone methyltransferases (HMTs) add methyl groups to lysine and arginine side chains. Histone demethylases remove the methyl groups Histone modifications are associated with gene regulation Acetylation is associated with active transcription - euchromatin has more acetylation than heterochromatin Acetylation removes the positive charge from lysine side chains, which affects interactions with negatively-charged DNA Histone acetylation also generates binding sites for bromodomain proteins, which bind specifically to certain acetyl-lysines Bromodomains then recruit other proteins like nucleosome remodeling complexes Up to three methyl groups can be added to a lysine, and up to two can be added to an arginine Methylation is associated with transcription activation or repression, depending on the residue that is methylated, so the position as well as the Chromosome structure and function Side notes: modification are significant for regulation Methylation of lysine 9 in the H3 tail is associated with silent chromatin Methylation of H3 lysine 4 is associated with active chromatin Chromodomains bind to specific methylated lysines, and are often associated with transcriptional silencing Nucleosome remodeling complexes control access to DNA Chromatin packaging presents a barrier to proteins that need to access DNA, and hence inhibits processes like transcription Nucleosome locations can be changed to allow proteins to access the DNA ATP-dependent nucleosome remodeling complexes increase accessibility of DNA by: Sliding the histone octamer along the DNA, or Removing the histone octamer and transferring it elsewhere, or Introducing transient loops into the DNA wrapped around a histone core Several classes of nucleosome remodeling complexes exist, with different subunit compositions, such as: SWI/SNF: disrupts nucleosome positioning ACF (ISWI family): positions nucleosomes during chromatin assembly Nucleosome remodeling complexes have subunits such as: ATPase domain (in all complexes) in two parts - Dexx and HELICc Bromodomain (binds acetylated lysines) Chromodomain (binds methylated lysines) SANT-SLIDE (binds histone tails) Nucleosome remodeling complexes can be recruited by transcription factors or histone modifications Nucleosome remodeling is required to be precise, to allow exposure of specific regions of DNA Cells recruit remodeling complexes via proteins that bind in a sequence specific manner Transcriptional regulators and histone modifications can recruit nucleosome remodeling complexes DNA can be methylated Methylation occurs in bacteria and many eukaryotes Methyl groups can be added to cytosine residues to make 5-methyl cytosine by DNA methyltransferases (aka DNA methylases) Eukaryotic methylation is usually at CpG or CpXpG sites (p means phosphodiester bond, X means any base) DNA methylation can lead to base changes DNA methylation is risky - DNA can be altered permanently Methylated cytosines are less chemically stable, so more likely to undergo deamination, which change the base to thymine This recognized as damage to the DNA and is repaired to restore the original cytosine DNA methylation plays roles in gene regulation Methylation in eukaryotes is used to silence transcription Up to 60% of human CpGs are methylated, and methylation state can be inherited Some DNA binding complexes bind specifically to methylated DNA, such as chromosome structure and function Side notes: histone modification enzymes and nucleosome remodeling complexes Similarly, some histone binding proteins can recruit DNA methyltransferases Centromeres direct chromosome segregation In eukaryotes, centromeres direct chromosome segregation Centromeres are found in all eukaryotic chromosomes and are necessary for correct chromosome segregation during cell division Most species have a single centromere per chromosome The centromere is in a heterorochromatic region, and is inherited After DNA replication, but before chromosomes separate, two sister chromatids are present. The centromere appears as a construction Centromeres bind specific proteins to form a structure called a kinetochore In mitosis, kinetochores attach to microtubules from opposite spindle poles This attachment lets the spindle pull the sister chromatids apart This mechanism of chromosome segregation is highly conserved, but centromere sequences in different species are distinct Centromere structure varies across species Centromeres vary in size and sequence in different organisms Some organisms (as in C. elegans) have no defined centromere, and spindles attach along the length of the chromosome Human centromeres are made of tandem repeats (alpha-satellite repeats) that are arranged in higher order repeats Centromeres all have a defined region where the nucleosomes have a histone H3 variant (CENP-A in humans) The CENP-A marked region is the domain where kinetochores assemble and accurate CENP-A deposition is crucial for correct centromere function Telomeres are defined by repetitive sequences Telomeres stabilize the ends of chromosomes and help solve the problem of replicating chromosome ends Telomere DNA consists of simple tandem repeats with one C-rich strand and one G-rich strand The G-rich strand in all eukaryotes is the strand that extends 5'-3’ towards the chromosome end and terminates in a small single-stranded region Human telomeres consist of repeats of TTAGGG; other organisms have different repeats The total length of the repetitive region ranges from 50 - 30,000 bp, depending on species Telomeres protect the ends of linear chromosomes The G-rich strand in all eukaryotes is the strand that extends 5'-3’ towards the chromosome end, and it terminates in a small single-stranded region In organisms with long telomeres, the overhang may be processed into a tloop, formed by base-pairing of the overhang with the double-stranded region Telomere repeats are binding sites for proteins that mark them as natural ends and distinguish them from DNA breaks (which might be inappropriately repaired) DNA polymerase cannot copy the ends of a DNA molecule - telomerase's maintain the ends of the chromosomes Chromatin packing has several layers chromosome structure and function Side notes: The first level is the 10 nm fiber, which arises from nucleosome association with DNA, and has a "beads on a string" appearance The 10 nm fiber is folded further into the 30nm fiber Formation of the 30nm fiber involves the linker histone H1 Chromatin compaction may reflect transcriptional activity In interphase, there is a range of compaction along a chromosome Chromatin in relatively decondensed regions stains lightly - euchromatin More compacted regions stain more darkly - heterochromatin Heterochromatin is associated with less transcriptional activity Telomeres, centromeres, and regions of highly repetitive DNA are typically located in heterochromatin Chromosomes are packaged hierarchally DNA is packaged into nucleosomes Nucleosome-bound DNA is then assembled into topologically associated domains (TADs) The chromosomes then occupy distinct chromosome territories in the nucleus The nuclear location of a chromosome is correlated with its transcriptional activity Chromosomes occupy specific territories in the eukaryotic nucleus Each chromosome occupies a characteristic space - a chromosome territory - as shown by chromosome fluorescence in-situ hybridization (FISH) staining The mechanism for establishing chromosome territories is not yet understood Nuclear location correlates with gene activity Heterochromatic regions are often associated with the nuclear periphery Human chromosomes 18 and 19 are of similar size - gene-rich 19 is found near the center of the nucleus, gene-poor 18 is at the periphery In female mammals, the inactive X chromosome is always at the nuclear periphery