The Chromatin & Epigenetics - Lesson 1: The Structure of Chromatin PDF

The chromatin & epigenetics Lesson 1 The structure of chromatin Zsolt Fábián M.D., Ph.D., Dr. Habil.  The chromatin & epigenetics - Lesson 1: The structure of chromatin TEM electronmicrograph of a human nucleus Credit: Profess...

The chromatin & epigenetics Lesson 1 The structure of chromatin Zsolt Fábián M.D., Ph.D., Dr. Habil.  The chromatin & epigenetics - Lesson 1: The structure of chromatin TEM electronmicrograph of a human nucleus Credit: Professor L. Komáromy Medical School, University of Pécs, Hungary  The chromatin & epigenetics - Lesson 1: The structure of chromatin DNA is bent by proteins IHF Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  The chromatin & epigenetics - Lesson 1: The structure of chromatin Histones ▪ There are four core histones (evolutionary conserved gene family) ▪ Rich in lysine and arginine ▪ Histones form the histone octamer – two copies each of the four core histones H2A, H2B, H3 and H4 ▪ The two H3-H4 dimers associate with DNA ▪ The two H2A-H2B dimers then associate to form the octamer ▪ About 146bp of DNA wraps around the octamer to form the nucleosome ▪ 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 Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  The chromatin & epigenetics - Lesson 1: The structure of chromatin Molecular structure of nucleosomes ▪ 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 Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  The chromatin & epigenetics - Lesson 1: The structure of chromatin Histone variants ▪ The four core histones are the most common ▪ Other histone variants can be incorporated into nucleosomes, and are found at special chromatin locations ▪ For example, H2A.X is phosphorylated at sites of DNA double-strand breaks and is thought to recruit repair machinery Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  The chromatin & epigenetics - Lesson 1: The structure of chromatin Morphological organization of the chromatin decondensed DNA 10 nm fiber of nucleosomes „beads-on-the-string” are associated with DNA structure Core nucleosome is an octamer of histone proteins solenoid structure wrapped with 1.8 turns of DNA (= 146 bp in length) Core nucleosome is 2 copies of each core histone: H2A, loose chromosome H2B, H3 & H4 a single H1 histone – is attached to edge of core – like a handle condensed chroosome Nucleosomes wrap up DNA (2nm) separated by ~50 bp of linker DNA to form "Beads-on-a-string" = “10 Mitotic chromosome nm fiber Based on Jansen A., Verstrepen K., J., Microbiol. Mol. Biol. Rev. (2011) 75(2):301-20  The chromatin & epigenetics - Lesson 1: The structure of chromatin The „beads-on-the-string” structure Credit: Professor L. Komáromy Medical School, University of Pécs, Hungary  The chromatin & epigenetics - Lesson 1: The structure of chromatin Morphological organization of the chromatin decondensed DNA „beads-on-the-string” structure The 10 nm fiber is folded further into the 30nm fiber, a regular solenoid structure arrangement that brings nucleosomes together loose chromosome condensed chroosome Mitotic chromosome Based on Jansen A., Verstrepen K., J., Microbiol. Mol. Biol. Rev. (2011) 75(2):301-20  The chromatin & epigenetics - Lesson 1: The structure of chromatin Solenoid DNA Credit: Professor L. Komáromy Medical School, University of Pécs, Hungary  The chromatin & epigenetics - Lesson 1: The structure of chromatin Solenoid structure ▪Formation of the 30nm fiber involves histone H1 ▪ H1 is a linker histone that binds to the linker DNA in between successive nucleosomes, helping compaction ▪ The core histone tails are also involved in formation of the 30 nm fiber, but it is not fully understood how. Based on Jansen A., Verstrepen K., J., Microbiol. Mol. Biol. Rev. (2011) 75(2):301-20  The chromatin & epigenetics - Lesson 1: The structure of chromatin Morphological organization of the chromatin decondensed DNA „beads-on-the-string” structure solenoid structure The 30 nm fiber is then compacted further into compact loose chromosome chromosomes in which large loops of chromatin are anchored to a central scaffold condensed chroosome Mitotic chromosome Based on Jansen A., Verstrepen K., J., Microbiol. Mol. Biol. Rev. (2011) 75(2):301-20  The chromatin & epigenetics - Lesson 1: The structure of chromatin Chromatin Condensation of DNA Reduce Size - DNA from all 46 chromosomes is appr. 2m vs. the 6μm of nuclear diameter Neutralize charge along the backbone Protect & control the DNA (& its code) Prokaryotes supercoiling into twisted loops linked to a very small RNA “core” coating with positively charged polyamines; spermine/spermidine Eukaryotes DNA is wrapping around small, positively charged histon proteins compaction into structured chromatin  The chromatin & epigenetics - Lesson 1: The structure of chromatin Mitotic chromosome Credit: Professor L. Komáromy Medical School, University of Pécs, Hungary  The chromatin & epigenetics - Lesson 1: The structure of chromatin Karyotyping ▪ Condensed chromosomes have characteristic lengths and banding patterns ▪ A karyotype is a display of the chromosomes from an individual, derived from microscopy images prior to separation of sister chromatids Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  The chromatin & epigenetics - Lesson 1: The structure of chromatin Chromatin regions with specific functions - centromere Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014) Credit for TEM: Professor L. Komáromy Medical School, University of Pécs, Hungary  The chromatin & epigenetics - Lesson 1: The structure of chromatin Chromatin regions with specific functions - centromere Human centromeres are about 1MB long and are made of repetitive sequences (alpha-satellite repeats) – tandem repeats of 171bp arranged in higher order repeat Centromeres all have a defined region where the nucleosomes have a histone H3 variant (called CENP-A in humans, other names in other organisms) CENP-A has specificity for centromeric regions, particularly AT-rich areas The CENP-A marked region is the domain where kinetochores assemble and therefore CENP-A is crucial for centromere function Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  The chromatin & epigenetics - Lesson 1: The structure of chromatin Chromatin regions with specific functions - centromere ▪ 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 Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014  The chromatin & epigenetics - Lesson 1: The structure of chromatin Chromatin regions with specific functions - telomere ▪ Telomere DNA consists of simple 50-30,000 bp long tandem repeats (TTAGGG in human) with one C-rich and one G-rich strand ▪ The G-rich strand extends 5′-3′ towards the chromosome end and terminates in a small single-stranded region in all eukaryotes ▪ In organisms with long telomeres, the overhang may be processed into a t-loop, 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) ▪ In humans, telomerase is active, and telomere length maintained, in stem and germline cells ▪ In mature tissues, there is often insufficient telomerase, so telomere shortening occurs that limit the number of cell divisions ▪ Over-activation of telomerase is implicated in many cancers as it allows cells to grow inappropriately ▪ Telomeres are heterochromatic Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  The chromatin & epigenetics - Lesson 1: The structure of chromatin Telomere-binding proteins ▪ Telomeres bind proteins that protect the ends and help maintain the length ▪ There are three classes of telomere binding proteins ▪ End-binding proteins protect the G-rich overhang ▪ Double-stranded binding proteins bind along the length ▪ Proteins that associate with the DNA binding proteins ▪ Telomere-binding proteins regulate length – overexpression results in shorter telomeres ▪ The unprotected end is “repaired” as a DNA break, which causes broken and aberrantly joined chromosomes – DNA information is then not stably heritable Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  The chromatin & epigenetics - Lesson 1: The structure of chromatin Summary Annunziato, A. (2008) DNA Packaging: Nucleosomes and Chromatin. Nature Education 1(1):26 The chromatin & epigenetics Lesson 2 Epigenetics Zsolt Fábián M.D., Ph.D., Dr. Habil.  The chromatin & epigenetics - Lesson 1: The structure of chromatin Summary Annunziato, A. (2008) DNA Packaging: Nucleosomes and Chromatin. Nature Education 1(1):26  The chromatin & epigenetics – Lesson 2: Epigenetics Chemical modifications of histones Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  The chromatin & epigenetics – Lesson 2: Epigenetics Chemical modifications of histones Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  The chromatin & epigenetics – Lesson 2: Epigenetics Histone acetylation level of acetylation varies considerably Histone Acetyltransferases (HATs) add acetyl groups euchromatin has more acetylation than heterochromatin acetylation is associated with active transcription 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 remodelling complexes Histone Deacetylases (HDACs) remove acetyl groups Histone deacetylation is associated with heterochromatin and gene silencing Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  The chromatin & epigenetics – Lesson 2: Epigenetics Histone methylation Methylation is associated with transcription activation or repression, depending on the residue that is methylated, so the position as well as the 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 Up to three methyl groups can be added to a lysine, and up to two can be added to an arginine Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  The chromatin & epigenetics – Lesson 2: Epigenetics Histone phosphorylation Phosphorylation at serine 10 promotes acetylation of lysine 14 Acetylation of lysine 14 inhibits methylation of lysine 9 ▪Phosphorylation provides additional regulatory signals ▪ Phosphates are added by kinases and removed by phosphatases -Phosphorylation of H3 serine 10 allows cell growth transcription -Phosphorylation of H3 serine 10 and serine 28 correlate with chromosome condensation in mitosis ▪ Modifications can positively or negatively affect modifications at other residues (see above) ▪ The wide range of modification possibilities, in addition to the interactivity of modifications, has led to the hypothesis of a histone code, in which unique modification combinations define certain chromatin states Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  The chromatin & epigenetics – Lesson 2: Epigenetics Nucleosome remodeling ▪ Chromatin packing 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 loops into the DNA wrapped round a histone core Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  The chromatin & epigenetics – Lesson 2: Epigenetics Nucleosome remodeling 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 (a) and histone modifications (b) can recruit nucleosome remodeling complexes Craig et al., Molecular Biology - Principles of Genome Function, 2e, Oxford University Press, (2014)  The chromatin & epigenetics – Lesson 2: Epigenetics The histone „code” - epigenetics ▪ Epigenetics - heritable changes in gene expression without changes to the DNA sequence Chromosome ▪ Epigenetic change is mediated by DNA methylation, histone modification and „Inactiv” heterochromatin non-coding RNA (ncRNA)-associated H3K9me3 gene silencing. H3K20me3 H3K27me3 ▪ Epigenetic modifications lead to the Nucleosome Histon creation of the ‘epigenome’ Histon modification Histon octamer ▪ The epigenome undergoes biochemical changes in response to environmental stimuli and leads to the remodeling of chromatin structure Nucleosome „Activ” euchromatin ▪ Increasing histone acetylation levels by Histon acetylation H3K4me3 DNA blocking the HDAC can cause an increase H3K36me3 H3K79me3 in memory storage. Credit: Diagenode corporation  The chromatin & epigenetics – Lesson 2: Epigenetics The histone „code” - epigenetics ▪ Increasing histone acetylation levels by blocking the HDAC can cause an Chromosome increase in memory storage. ▪ Children born during the period of the Dutch famine in 1944-1945 have „Inactiv” heterochromatin increased rates of coronary heart H3K9me3 H3K20me3 disease and obesity after maternal H3K27me3 exposure to famine during early Nucleosome pregnancy. Histon Histon modification ▪ Less DNA methylation of the Histon octamer insulin-like growth factor II (IGF2) gene is associated with this exposure. ▪ Likewise, adults that were prenatally Nucleosome exposed to famine conditions have „Activ” euchromatin Histon acetylation also been reported to have H3K4me3 DNA H3K36me3 significantly higher incidence of H3K79me3 schizophrenia. Credit: Diagenode corporation  The chromatin & epigenetics – Lesson 2: Epigenetics Methylated DNA Bases 5-Methylcytosine N6-Methyladenosine No changes in base-pairing properties Eukaryotes: only methylcytosine seems important Bacteria: both methylcytosine and methyladenosine Only C's followed by G's can be methylated – 60-90% of CpG's are methylated in the human genome Promoted by methylase enzyme families in all cells – Recognition sites are palindromic (see upcoming topics)  The chromatin & epigenetics – Lesson 2: Epigenetics Methylated DNA Bases - Functions Bacteria Controlling initiation of replication Discrimination of self DNA (methylated) from foreign DNA (non-methylated) Discriminate old & new strands in mismatch repair Sometimes involved in regulation of gene expression Eukaryotes Normal regulation of chromatin structure & gene expression – More extended & stabilized = “Gene silencing” – An epigenetic mechanism of inheritance e.g. Imprinting and X-Chromosome inactivation  The chromatin & epigenetics – Lesson 2: Epigenetics Epigenetics Epigenetics studies of changes in inheritable gene function that do NOT entail a change in DNA base sequence. Epigenetics is mediated through mechanisms that do not directly depend on a specific DNA sequence, e.g., switching between heterochromatin and euchromatin to silence or activate a gene Epigenetics is dynamic (= cell response to regulatory signals) – OR – stabile (passed down from ancestral cells over several generations or just during a cell’s lifetime) The chromatin & epigenetics – Lesson 2: Epigenetics The histone „code” - epigenetics  The chromatin & epigenetics – Lesson 2: Epigenetics Medical relevance of epigenetics - Rubinstein-Taybi syndrome ▪ CBP is a well-known transcriptional coactivator that has HAT activity and has been shown to be important for long-term memory formation. ▪ Mutations in CBP can contribute to the pathology of Rubinstein-Taybi syndrome, a neurodevelopment disorder characterized by cognitive impairment, short stature, broad thumbs and first toes, and characteristic facial features, such as microcephaly. Bartsch et al., Journal of Medical Genetics (2002) ;39:496-501. The chromatin & epigenetics – Lesson 2: Epigenetics Summary