BIOB11 - Lecture 7
THE HISTONE CODE: acetylation of N terminal tail = activation; methylation of N terminal tail = silencing. *postulates that the state and activity of a particular region of chromatin/DNA depends on the specific chemical modifications of various enzymes (deacetylases, methylation etc. - acetylation of lysine leads to transcriptional activation or 'unwraps' the chromatin fiber)*. 1. modified residues serve as docking sites to recruit a specific array of non-histone proteins, which then determine the properties and activities of that segment of chromatin. 2. modified residues alter the manner in which the histone tails of neighbouring nucleosomes interact with one another or with the DNA to which the nucleosomes are bound. - The N-terminal tails of histones project outwards from the core - Several enzymes can modify these tails by phosphorylation, acetylation etc. (histones can be unwrapped with histone acetyl transferase to access DNA of interest) - histonedecaetylase is used to re-wrap once transcription is done - The pattern of such modification influences the properties of nucleosomes and in turn, regions of chromatin epigenetic - heritable chemical changes to histone proteins my notes: 'The histone code is a hypothesis that the transcription of genetic information encoded in DNA is in part regulated by chemical modifications to histone proteins, primarily on their unstructured ends. Together with similar modifications such as DNA methylation it is part of the epigenetic code.[1] Histones associate with DNA to form nucleosomes, which themselves bundle to form chromatin fibers, which in turn make up the more familiar chromosome. Histones are globular proteins with a flexible N-terminus (taken to be the tail) that protrudes from the nucleosome. Many of the histone tail modifications correlate very well to chromatin structure and both histone modification state and chromatin structure correlate well to gene expression levels. The critical concept of the histone code hypothesis is that the histone modifications serve to recruit other proteins by specific recognition of the modified histone via protein domains specialized for such purposes, rather than through simply stabilizing or destabilizing the interaction between histone and the underlying DNA. These recruited proteins then act to alter chromatin structure actively or to promote transcription. For details of gene expression regulation by histone modifications see table below.'
*read in textbook
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1. Growth (Interphase) A. G1 - cell growth and metabolic activity B. S - DNA replicates and produces two copies of each chromosome C. G2 - cell continues to prepare for mitosis/cell division 2. Mitosis (Cellular Division) (M phase) A. prophase - chromosomes become visible and condensed, each identical copy of single chromosome is called a sister chromatid. Nuclear envelope breaks down and spindle fibres form, microtubules grow out of centrioles migrating to opposite poles B. metaphase - double stranded chromosomes line up on either side, Mts attach to each sister chromatid C. anaphase - when chromatids begin to separate, centromere divide and the chromosomes move away from each other along spindle finer D. telophase - two groups of chromosomes reach opposite ends of cell, new envelope begins to form 3. Cytokinesis (Cytoplasm division) - division of cells resulting from mitosis, produces 2 genetically identical cells
Briefly describe the cell cycle.
The process by which DNA is copied to RNA is called transcription, and that by which RNA is used to produce proteins is called translation. DNA REPLICATION Each time a cell divides, each of its double strands of DNA splits into two single strands. Each of these single strands acts as a template for a new strand of complementary DNA. As a result, each new cell has its own complete genome. This process is known as DNA replication. Replication is controlled by the Watson-Crick pairing of the bases in the template strand with incoming deoxynucleotide triphosphates, and is directed by DNA polymerase enzymes. It is a complex process, particularly in eukaryotes, involving an array of enzymes. A simplified version of bacterial DNA replication is described in Figure 2.
Describe DNA transcription, translation, and replication.
Maintenance of the 30nm fiber depends on interactions between histone molecules of neighbouring nucleosomes - Histone tails are required for this condensation into the 30nm fiber - *chromatin* is formed of packed 6 nucleosomes (167 bp DNA and histone octamer) https://www.youtube.com/watch?v=bwVjYxcDQ5I - The fiber is packaged roughly 6 nucleosomes per turn w/ H1 facing the inside of the solenoid structure - Chromatin regions that are not being actively transcribed are present mostly in either 30nm fibers or even higher ordered structures (looped domains) (*heterochromatin*) - 30 nm fibers can be seen under Electronic Microscope at physiological [salt] conditions *looped domains*: looks like a flower - https://www.youtube.com/watch?v=TR2ojQczEQY 30 nm fiber may be further gathered into a series of large, supercoiled loops/domains to form even thicker fibers 80-100nm - these loops are attached to nuclear proteins that are part of the nuclear matrix (scaffold) ➢ eg. Topoisomerases - orally ot visile eause they're spread out - can be visualized by removing histones form mitotic chromosomes - cohesion forms a ring on the fiber and helps form loops holds loops together
Describe chromatin, and describe looped domains.
PIGENETICS - Inheritance that is not based on DNA seq. alone - *Covalent modifications of DNA (eg. Methylation), histones, and other types of chromatin modifications may be the carriers of this epigenetic information ➢ X-chr. Inactivation is an example* ➢ The histone code may be critical in determining the heterochromatin regions in offspring eg. Histone modification are somehow inherited and replicated from parent to offspring
Describe epigenetics in one sentence, and provide an example.
At stem cell stage, orange gene became a Barr body and all cells expressed instead produced other X chromosome (black pigment). EPISTASIS results in production of white pigment (tell us if the colour/pigment orange or black is actually added to the hair or not) so we see white sometimes in addition to orange black. We can only rarely find XXY calico cats (male). Likely occurs through methyl deacetylation of N terminal domain. *class notes*: X-CHROMOSOME INACTIVATION - Occurs early during embryonic development in female mammals - Random process where either maternal or paternal X-chr. Is inactivated in any given cell - Random inactivation makes adult female genetic mosaicsbut, both X-chr. Are active in germ cells prior to meiosis ➢ Eg. Calico cats are usually female
Explain barr bodies/facultative chromosome inactivation with an example. (This FC and slide)
- Histone H1 + core nucleosome form highly compact 30nm fibers that increase the DNA packaging ratio 6 fold further (now 40fold) - Nucleosomes line up end-to-end into 2 stacks of nucleosomes that form a double helical structures ➢ H1 histones form a solenoid structure and nucleosomes line up to form helical stacks
Explain the 30nm fiber/highest level of chromatin structure (answer on slide/FC)
Histone itself is made up of multiple *histone subunits*. H2A, H2B, H3, H4. Are arranged into a histone 'bead' which allows the DNA structures to wrap themselves or coil around this bead, ultimately compressing it for DNA storage.
Explain the structure of histones as a nucleosome core particle.
cells prepare for mitosis - looped domains get further compacted into chromosomes. DNA packing ratio in chromosome is 10K fold. (10 micrometers nucleus can pack 200k times this length of DNA). pencil analogy. Book: chromatin loops are attached at their bases to a residual protein scaffold, and linked together at their base with a cohesin ring.
How do cells prepare genetic material for meiosis?
Non-specific nucleases (DNAse 1) cuts into 200 bp length fragments with nucleosomes acting as linkers to ensure even fragments (treating naked DNA with DNAse 1 instead creates random fragments, these act as 'dividers')
How does cleavage of DNA coiled around histones occur? Why?
The N terminal segments from each histone assume the form of a long, flexible tail, and extend out *through* the DNA helix that is wrapped around the core particle. For many years, histones were thought of as inert structural molecules, but the extending N terminal segments play a role in making the chromatin accessible to proteins. In this way, chromatin is a dynamic cellular component in which histones, regulatory proteins, and a variety of enzymes move in and out of the nucleoprotein complex to facilitate the complex tasks of DNA transcription, compaction, replication, recombination, and repair.
Is chromatin a static, silenced form of DNA preservation or something more dynamic?
Importantly this occurs only in dividing cells, and allows miles of DNA to be compactly preserved in chromosomes for assignment during meiosis II. Starts with assembly of nucleosome, 8 different H1 histone protein subunits attach to DNA and the DNA winds along it to form the nucleosomes, which then stack on top of each other, forming a finer known as chromatin (*chromatosome*). This fibre is 30 nm in thickness, and is then looped and further packaged using even more additional accessory proteins. Lets 6 feet of DNA fit into the nucleus (so small that 10,000 *class notes* PACKAGING THE GENOME - Genome must be packaed in the nucleus in order to fit ➢Diploid cell has 6.4b bp of DNA in 46 chr.length of bp = 0.34 nmabout 2m of DNA • Also binds to 6 water molecules expands volume • All has to fit in a 10 micrometer nucleus - Packaging must be compact, but DNA still needs to be accessible to enzymes and regulatory factors - DNA + ProteinsChromatinforms chromosomes in dividing cells - 2 Types of Proteins 1. Histones: most abundant, small basic proteins (high in Lys/Arg, w/ +ve charge) 2. Non-histone proteins: diverse structural, enzymatic, and regulatory proteins
VERY good 2 min video https://www.youtube.com/watch?v=eYrQ0EhVCYA
Histone modification affects degree of compaction of euchromatin into heterochromatin (i.e., by methylation or deacetylation of the N terminal tail and thereby silencing the gene); its ability to interact with neighbouring histones via its N terminal tail; and the likelihood of its transcription (i.e., when histone acetyltransferase or HAT comes in it ACTivates the gene by 'unwinding' it from the nucleosome core particle (167bp segment wrapped around histone octamer and H1 linker; rendering it vulnerable to transcription by RNA polymerase).
What 3 things does histone modification affect?
HISTONES - Small, basic proteins rich in basic amino acids Lys, Arg - give protein high +ve charges - can easily interact w/ -ve charge of DNA - 5 Major types/classes depending on Arg/Lys ration - highly conserved since they either interact w/ DNA or other histonesmost amino acids cannot be replaced ➢ mutations are generally not tolerated - can be modified by phosphorylation and acetylation (post-translationally)
What are histones?
➢ Remove H1 w/ low [salt] solution (low ionic strength) ➢ Digest DNA w/ non-specific nuclease ➢ Remove core histones w/ high [salt] solution • See ~200bp fragments of DNA after non-specific nuclease digestion - Process: see that linker DNA is digested, after this add an high [salt] solution to remove all histonesrun on gelroughly 200 bp determined that there was a roughly 200bp repeating structure
What experiment can we do to determine the presence of histones? Compare to above slide from Shomu!**
- At interphase, after the completion of mitosis, most DNA becomes less compacted and is functionally activecalled Euchromatin 10% always remains compacted and has little to/no functional activity (called heterochromatin) ➢ is on edges, more tightly packed
What happens after mitosis? Explain in this and next FC.
https://www.youtube.com/watch?v=Y9vXhmI5FXM once we begin to have stem cells in gametes (fused in embryo) forming a blastula, a coin flip occurs in each stem cell and one of the 2 x chromosomes is silenced; but can be either one that is activated in each cell. for the rest of the life of the cell and all its progeny, only that x chromosome is expressed. this explains why calico cats look the way they do! black hair gene on 1 x chromosome, orange on the other.
What is a Barr body and how does it explain facultative heterochromatin?
*euchromatin* = U, looped and loose. *hetero* densely packed/supercoiled - phallic. wrapped around histones and non-functional. divided into *constitutive* (most of the heterochromatin, perform structural role like centromeres and telomeres - a constitutive heterochromatin section - controls expression of certain adjacent genes) and *facultative* heterochromatin (those which are completely worthless - silenced by histone deacetylation or methylation, or some other form of events). both types are found surrounding the nucleus on the outside (like a 'heater' fertile egg) *euchromatin* - FUNCTIONAL - open for access, no histones - free for transcription, replication, etc. some are attached with histones (but not supercoiled) - only in nucleosomes with linker regions) - sometimes facultative can be turned back under genetic regulation (using histone chemical modification) and rendered accessible (u chromatin) - using histone acetylation of the N terminal tail we can 'unwind' the euchromatin comprising nucleosomes and linker regions into something more resembling euchromatin *without* histone octamer. https://www.youtube.com/watch?v=v8CzEg-SiqM
What is the difference between heterochromatin and euchromatin? (Answer on slide) Explain the two types of heterochromatin and their role, where they are found (answer on this and next slide, FC)
• H1 is essentially binds the DNA together - Experiment:
What is the main function performed by H1?
- Dimer formation is mediated by CTA (alpha helices) - N-terminal forms long, flexible tail that extends outward
What is the role of the C terminal domain in dimer formation?
In addition to binding to the nucleosome, the H1 protein binds to the "linker DNA" (approximately 20-80 nucleotides in length) region between nucleosomes, helping stabilize the zig-zagged 30 nm chromatin fiber. H1 links nucleosomes into higher order structures.Book: Maintenance of the 30nm finer depends on interactions between histone molecules of neighbouring nucleosomes. Linker histones and core histones have both been implicated in higher order packaging of chromatin. For example if H1 linker histones are selectively extracted from compacted chromatin, the 30 nm fibres uncoil to form the thinner, more extended beaded filament - adding back H1 histone leads to restoration of the higher order structure. Core histones of adjacent nucleosomes may interact with one another by means of their long flexible tails (N terminal tail can associate with neighbouring histones). This is compounded by the negatively charged phosphate backbone of DNA and the positively charged histone molecule covered in amino acid residues, which results in greater adhesion overall. *Looped domains* are held together by the protein *cohesin*.
What is the role of the H1 histone?
*A model in which small RNAs govern the formation of heterochromatin*: recent studies suggest that non-coding RNAs play a role in heterochromatin formation. 1. RNAs are transcribed from both strands of repetitive DNA sequences 2. form double stranded molecules that are 3. processed by Dicer and other components 4. siRNA protein complex binds to complementary segment of nascent RNA and recruits histone methyltransferase, methylating the H3 histone's K9 residue, replacing acetyl groups (wrapped), and 5. these methyl sites act replace all acetyl groups and 6. act as 'docking' regions for HP1 protein 7. DNA boundary element prevents spread of heterochromatinization into neighbouring regions and instead facilitates supercoiling of chromatin in these specific regions. 8. SUV39H1 enzyme can also bind to facilitate additional methylation 9. a region of highly compacted heterochromatin has been formed (see blue segment on slide) from *class notes*: Small RNAs like siRNAs, initiate the process by which methylation of euchromatin ultimately leads to hetrochromatin formation and DNA compaction - Acetylation allows for euchromatin to formb - Hp interacts w/ other Hp units and makes DNA even more compact Process: repeated DNA is transcribedcreate a dsRNA sliced by dicer to create siRNAinitiate process of removing acetyl groups and adding methyl groups through help of SUV39H1HP1s add on to methyl groupincreased compaction - histone modification may affect ➢ degree of compaction (eg. Hetero/euchromatin) ➢ likelihood that a gene is transcribed ➢ alter interactions w/ histones and non-histone proteins eg. Removal of acetyl groups from H3 and H4 is among the initial steps in conversion of euchromatin into heterochromatin eg. Lys in position #9 of H3 when methylatedformation of heterochromatin ➢ in euchromatin, the same residue is acetylated instead ➢ H4 is de-actelyedH3 (Lys9) is methylatedthis methylated K9 can bind specific proteins containing chromo domains • Eg. Heterochromatic protein 1 (HP1)HP1 binds to other HP1 proteins on neighbouring nucleosomesmore compacted DNA
steps in forming discrete regions of heterochromatinization (difficult to wrap head around, read a few times then try relaying back)
- Small RNAs like siRNAs, initiate the process by which methylation of euchromatin ultimately leads to hetrochromatin formation and DNA compaction - Acetylation allows for euchromatin to formb - Hp interacts w/ other Hp units and makes DNA even more compact Process: 1. repeated DNA is transcribed 2. create a dsRNA 3. sliced by dicer to create siRNA 4. initiate process of removing acetyl groups and adding methyl groups through help of SUV39H1HP1s 5. add on to methyl group 6. increased compaction
write FC in own words
