BMS 532.09 Chromosome Structure and Function.pptx

Chromosom e Structure and Function A N E X P LO RAT I O N O F D N A O R G A N I Z AT I O N W I T H I N A N ORGANISM BMS 532 B LO C K 2 L E C T U R E 2 Objectives: General/Background 1. Define the following terms: chromatin, chromosome, chromatid, chromosome territories, histone, nucleosome, cent...

Chromosom e Structure and Function A N E X P LO RAT I O N O F D N A O R G A N I Z AT I O N W I T H I N A N ORGANISM BMS 532 B LO C K 2 L E C T U R E 2 Objectives: General/Background 1. Define the following terms: chromatin, chromosome, chromatid, chromosome territories, histone, nucleosome, centromere, kinetochore, neocentromere telomere, karyogram, karyotype, autosome, repetitive DNA, pseudoautosomal regions, banding/banding pattern, euchromatin, heterochromatin, locus (loci), and allele. Chromosomes Structure and Function 2.Explain the relationship between types of DNA sequences and the structures formed in chromosomes and identify the expected chromosome structure formed from a sequence of DNA or histone variant incorporated at a given chromosome position 3.Summarize the processes of chromosome condensation with particular emphasis on the components involved in the generation of fully condensed chromosomes and Assess the consequences for changes in protein availability or incorporation in terms of chromosome structure or condensation level 4.Summarize the function, location, gene content, and importance of the following chromosome structures: centromere, kinetochore, Objectives: Chromosomes Structure and Function (continued) 5. Explain the role of the centromere and kinetochore in chromosome segregation with an emphasis on components required for function and the role of centromere positioning to chromosome segregation 6. Connect the kinetochore, the processes of chromosome condensation, and centromere positioning with cellular regulation, kinase activity, and the Anaphase Promoting Complex from block 1 7. Assess the consequences for the chromosome or cell particularly in terms of chromosome integrity or DNA repair induction following changes in length, availability or activity of telomere/telomerase associated proteins, activity or function of telomerase 8. Evaluate the process of telomerase-mediated elongation of telomeres and Explain the role of telomere associated proteins in telomere maintenance and/or telomerase activity Nomenclature Section: Introductory level ONLY; no complex or difficult designations/rearrangements should be expected 9. Explain how chromosome abnormalities are named and identify the proper designation for a given change ◦ Be able to identify the specific change from a summary karyotype ◦ Be able to write the summary karyotype for a given specific change Key Terminology and DNA Sequences OBJECTIVE 1: D E F I N E T H E F O L L O W I N G T E R M S : C H R O M AT I N , C H R O M O S O M E , C H R O M AT I D , C H R O M O S O M E T E R R I T O R I E S , H I S T O N E , N U C L E O S O M E , CEN TROM ERE, KINETOCH ORE, N EOCENTROM ERE TELOM ERE, K A RYO G R A M , K A RYO T Y P E , AU T O S O M E , R E P E T I T I V E D N A , P S E U D O A U T O S O M A L R E G I O N S , E U C H R O M AT I N , H E T E R O C H R O M AT I N , LOCUS (LOCI) , AND AL L ELE. LO1 Terminology: Chromosomes vs Chromatid Chromatin ◦ Lesser condensed form of DNA with associated proteins ◦ Chromatin makes up a chromosome Chromosomes ◦ DNA molecule with associated proteins in more condensed form ◦ Contains key structures (centromere and telomeres) ◦ ANY fully condensed form even AFTER anaphase = a CHROMOSOME Chromatid ◦ Longitudinal subunits produced by chromosome replication Chromatin Remodeling Complex ◦ Protein aggregates that reorganize the LO1 Even More Terminology: Chromatin Structure Satellite DNA = highly repeated non-coding DNA sequences Centromere = attachment site for 2 sister chromatid Telomere = repeat DNA sequences on ends; sacrificial buffer Covered in next lecture: Heterochromatin = compact, heavily staining chromosome regions rich in satellite DNA and low in gene content Euchromatin = less condensed LO1, Types of DNA in LO2 Human DNA Unique Sequence ◦ Most common (~75%) ◦ Represented only once in a haploid set of chromosomes ◦ Genes that encode for proteins are of this type Repetitive DNA ◦ Highly repetitive sequence (>105 copies) ◦ Middle repetitive (102-104 copies) ◦ Interspersed repeated DNA LO1, LO2, LO4 Repetitive DNA Clustered repeated sequences ◦ Sequences are repeated in clusters in one or a few locations ◦ Generally heterochromatic ◦ ~10-15% of genome ◦ Arrays of tandem repeats ◦ Satellite DNA = collective name for these tandem repeat clusters (i.e. α-satellite DNA @ centromere) LO1, Interspersed LO2 Repetitive DNA Interspersed repeated sequences ◦ Mixed in with other elements throughout the genome ◦ SHORT ◦ Alu Family (a type of Short Interspersed Nuclear Elements (SINE); ~300 bp in length; related but not identical in sequence; ~10% of human DNA) ◦ Enriched in regions rich in GC content (G- light bands) ◦ LONG ◦ Long Interspersed Nuclear Element (LINE) ◦ predominantly L1 sequences ◦ up to 6 kb in length; ~20% of genome ◦ Enriched in regions rich in AT content (G- dark bands) LO1, LO2 DNA Organization and Structure in Cells: Chromatin and Chromosomes In the nucleus of a nondividing cell, chromatin fibers form discrete chromosome territories Chromosome territories are correlated with gene densities Territories of chromosome domains that are relatively gene rich tend to be located toward Schematic of the interior of the nucleus chromosome territories formed by 30-nm chromatin fibers within the nucleus of a nondividing cell. LO1 Human Chromatin LO1 Important Terminology Distinctions Karyogram – The pictorial representation of the chromosomes by size and other differentiating markers Karyotype – The summary information from a karyogram; describes numbers and appearance under a light microscope LO1 Human Normal Karyograms Karyotyp es: LO1 A few (potential) reminders… Linear chromosomes consist of linear sequences of genes – genetic information which specifies the physical expression of a phenotypic trait Homologous chromosomes contain the same sequence of genes which may vary in DNA sequence (alleles) and/or expression The specific position on a linear chromosome for a particular gene or sequence = locus (plural = loci) This designation is based on banding patterns established by cytogenetics X and Y chromosomes – sex chromosomes which are non-identical but share some genes (regions of homology; pseudo-autosomal regions) – Males are genetically haploid for most genes on the X chromosome which results in unique pattern of X-linked inheritance Chromosome Structure and Condensation HIGHER ORDER STRUCTURES FOR DNA = DNA + PROTEINS LO1, LO3, LO4 Chromosome (Chromatin) Structure Chromatin = stable, ordered complex of DNA and proteins (histones) ◦ Histones = major class of small proteins; highly conserved across organisms The nucleosome is the basic structural unit of chromatin Each nucleosome is composed of: ◦ a core particle = histone octamer (2 of each: H2A, H2B, H3, and H4) with ~~145 bp of DNA ◦ ~55 base pairs of DNA called linker DNA that links adjacent core particles and ◦ one molecule of histone H1 that binds to the core particle and to the linker DNA LO1, LO3 Chromosome Condensation Nucleosomes coil to form higher order DNA structure ◦ 30 nm fiber: a left-handed superhelix or solenoidal supercoil; contains 6 nucleosomes per turn 30 nm fiber condenses into compact metaphase chromosome in which DNA/histone complex is attached to scaffold of non- histone proteins CHROMOSOME LO1, LO3 STRUCTURES AND ORGANIZATION DNA double helix Beads on a string (Nucleosomes loosely arranged) 30nm Fiber (Nucleosomes tightly packed) Condensation of packed nucleosomes LO1, LO3 LO1, LO3 LO1, Structures: Centromere and LO2, LO4, LO5 Kinetochore The centromere is essential for chromosome segregation The centromere is a region of highly specialized chromatin that is largely heterochromatic and contains lots of alpha satellite DNA Centromere Proteins: there are more than 20 now known The primary function of the centromere is to be the foundation for the formation of the kinetochore Kinetochore ◦ Protein complex essential for proper chromosome segregation during mitosis ◦ Massive complex requires several proteins LO1, LO4, LO5, LO6 Centromeres Centromeres may be located in different regions of a chromosome: Metacentric = located in middle of chromosome Submetacentric = located closer to one end of chromosome Acrocentric = located near one end of chromosome Telocentric = located at the telomere LO5, LO6 LO1, LO3, Centromere and LO4, LO5 Histone Variants To form key structures in chromatin and chromosomes, a combination of DNA sequences and proteins are needed Centromeres have a combination of repetitive DNA and key histone variants CENP-A = histone H3 variant ◦ Found exclusively at functional centromeres ◦ Helps form the structures necessary for formation of the kinetochore Inappropriate or unexpected incorporation of centromeric histones to regions of DNA not intended to serve as a centromere forms a centromere = Neocentromere  The Centromere and LO1, Kinetochore LO4, LO5, LO6 LO1, LO4, More than LO5, 100 LO6 proteins are now known to participate in this complex structure The “heart” of the kinetochore is a specialized nucleosome containing CENP A LO4, LO5, LO6 Kinetochore: Another Perspective The kinetochore is a highly conserved structure Part of the structure holds the microtubule while other components are regulatory Kinases and phosphatases play a critical role in regulating the process particularly in regards to microtubule attachment ◦ Aurora B kinase ◦ CDK1  Connecting the LO4, LO5, Kinetochore with LO6 Prevention of Erroneous Cell Division As hinted at in Block 1, inappropriate attachment is monitored by a different mechanism than separation Erroneous attachments are eliminated by an poorly understood mechanism that likely relates to tension on the kinetochore and the activity of Aurora B kinase and chromosome oscillation ◦ This is an interesting area of research with much that remains to be understood LO1, LO4, LO7 The End-Replication Problem: Introduction to Telomeres The need for RNA primers and the limitations of DNA polymerases mean that not all parts of linear chromosomes can get replicated The ends of linear chromosomes cannot be fully replicated leading to progressive loss of information (End Replication Problem; More on this later) Telomeres = Repetitive sequences of DNA at the ends of the chromosomes that assist with protecting the chromosome from genetic loss (along with other critical functions) ◦ TTAGGG Sacrificial Buffer for Protection of Genetic Material Can be maintained or restored via an enzyme called telomerase  as LO1, LO4, LO7 Protective Caps Sufficiently long telomeres are able to protect the ends of chromosomes vis capping The telomere cap forms when telomeres with their associated proteins loop into themselves ◦ A 3’ overhang called the G-strand Overhang folds back and invades the dsDNA forming T and D loops ◦ The looped “cap” protects the 3’ overhang ◦ Associated proteins assist with this ◦ TRF 1 = telomere repeat binding factor 1 ◦ Complex with it = Length regulation ◦ TRF 2 = telomere repeat binding factor 2 ◦ Complex with it = Protective end cap Loss of capping = reduced chromosome integrity ◦ Critically short telomere is associated with cellular senescence and DNA repair triggers at G1 LO4, LO7, LO8 Figure 03.25: The function of telomerase. LO4, LO7, LO8 Telomerase Maintains Telomere Length 3’ TTAGGG TTAGGGTTA AATCCC AUCCCAAUC 5’ 3’ telomerase 5’ Elongation of 3’ end 3’ TTAGGG TTAGGG TTAGGGTTA AATCCC AUCCCAAUC 5’ 3’ telomerase 5’ Extension of lagging strand 3’ TTAGGG TTAGGG TTAGGGTTA AATCCC CCCAATCCC 5’ DNA polymerase LO4, LO7, LO8 Telomerase Telomerase is an enzyme responsible for telomere maintenance Telomerase has two main components: ◦Reverse transcriptase protein subunit (hTERT) ◦RNA component (hTR or hTERC) Telomerase associates with ~39 proteins hTERT hTR LO4, Role of Telomerase in Cell Survival: LO7, Diseases Associated with Mutations in LO8 Telomerase TERT = telomerase reverse transcriptas e TERC (or hTR) = human telomerase template RNA Garcia K et al. Nuc Acid Res 2007;35:7406-7416 LO1, LO4 Chromosome Structures: Nucleolar Organizer Regions (NORs) Located on the satellite stalks of acrocentric chromosomes Location of nucleoli formation in interphase Site of ribosomal RNA genes and production of rRNA Chromosome Analysis and Nomenclature THE L ANGUAGE OF CHROMOSOMES: IDENTIFICATION OF ANOMALIES AND MAPPING OF POSITIONING How do we talk about chromosomes? Picture a bird ◦ What type of bird are you imagining? ◦ Is everyone thinking of the same bird? ◦ What if every time you tried to talk about your specific bird, the person you were talking two was thinking of a totally different bird? ◦ To address this issue, there is an international standard for chromosome designations and conversations = Nomenclature LO2, LO4,LO 9 Human Chromosome Classification: A tale of the centromere Metacentric ◦ Large = 1 to 3 ◦ Short = 19 and 20 Submetacentric ◦ Large = 4 and 5 ◦ Medium = 6 to 12 and X ◦ Short = 16 to 18 Acrocentric ◦ Medium = 13 to 15 ◦ Short = 21, 22, and Y LO1, LO9 General Concepts for Chromosomes and Bands Banding resolutions and patterns vary depending on method p (short) and q (long) arms Arms are divided into regions ◦ Ex) chromosome 7 arm q has 3 regions: 7q1, 7q2, and 7q3 Regions are further divided into bands ◦ This is where the nomenclature of 7q34 as 7q three-four (not thirty-four) comes from Bands Contain ~5-10 Mb of DNA Gene content in bands is variable and represents functionality Final reports must state the level of banding resolution and method due to the variability in both LO9 Banding and Technique Staining Techniques Subclassification Description Q-banding Quinacrine dihydrochloride Similar to G-banding great for centromeric regions of 3, 4, & 13; some acrocentric chromosomes and the Y A-T rich fluoresce brightly QF Q bands by fluorescence QFQ Q bands by fluorescence using quinacrine QFH Q-bands by fluorescence using Hoechst 33258 dye G-banding Giemsa and trypsinization to partially digest chromosome A-T rich stain dark GT G-bands by trypsin GTG G-bands by trypsin using Giemsa GTL G-bands by trypsin using Leishman stain GTW G-bands by trypsin using Wright stain C-banding Barium hydroxide treatment followed by Giemsa Constitutive heterochromatin close to centromere and long arm of Y Dicentric chromosomes and variations of constitutive heterochromatin CB C bands by barium hydroxide LO9 Banding and Technique Staining Techniques Subclassification Description Continued R-banding Heated in a phosphate buffer and stained to produce the reverse of the G-banding RF R-bands by fluorescence RFA R-bands by fluorescence using acridine orange RH R-bands by heating RHG R-bands by heating using Giemsa RB R-bands by BrdU RBG R-bands by BrdU using Giemsa RBA R-bands by BrdU using acridine orange DAPI 4’,6-diamidino-2-phenylindole (DAPI)-staining Typically a counterstain in fluorescence microscopy Characterization of AT-rich (+ stain) or AT-poor (- stain) heterochromatic regions especially when counterstained with chromomycin A3 (preferentially binds to GC-rich) DA-DAPI DAPI-bands by Distamycin A and DAPI NOR Nucleolus organizing region staining Silver nitrate (accumulates in the NORs) T-banding Giemsa technique that stains telomeres and centromeres LO9 Karyotyping Basic Rules ◦ ORDER = Chromosome number, Sex chromosome complement, abnormalities ◦ Exception = triplet abbreviation prior to chromosome number (i.e. mos) ◦ Sex chromosome abnormalities are listed before autosomal with X before Y ◦ Autosomal = numerical order ◦ Multiple structural changes of homologous chromosomes are listed alphabetically via abbreviation (i.e. del before ins) ◦ Numerical precede structural for the same chromosome ◦ Letters or triplets are used to specify structurally altered chromosomes ◦ Parentheses = chromosomes involved (separated by semicolon) ◦ Second parentheses = exact band involved for first set of chromosomes (separated by semicolon) ◦ Aberrations are listed with X first and then in numerical order unless there is a three- break rearrangement (i.e. some insertions) then the receptor chromosome is listed before the donor ◦ Semicolons between chromosomes and breakpoints if 2 or more chromosomes altered; no semicolon in the second set of parentheses for rearrangements involving a single chromosome ◦ A break suspected at the interface of 2 bands should be assigned the higher band number or the number more distant from the centromere ◦ Different clones or cell lines are separated by a single slant line ◦ Square brackets after karyotype string to indicate number of cells of each line or clone ◦ Critical in cancer studies because of the potential for clonal evolution of the cells Abbreviations pter or Description p arm or q arm telomere LO9 qter add Additional material of unknown origin del Deletion or loss of chromosome material (terminal or interstitial) der Derivative or structurally rearranged chromosome with an intact centromere dic Dicentric chromosome dup Duplication hsr Intrachromosomal homogeneously staining region indicating gene amplification ins Insertion inv Inversion (peri or para) i Isochromosome mos Mosaic r Ring chromosome rec Recombinant chromosome due to meiotic crossing- over t Balanced translocation Trc Tricentric chromosome LO9 Abbreviated/Short vs Detailed Short system rules are retained in the detailed system Exception: ◦ An abbreviated description of band composition starting at pter and progresses to qter replaces breakpoints within the last parentheses ◦ Bands are identified in order of occurrence within the derivative chromosome ◦ Single colon = break ◦ Double colon = breakage and reunion ◦ Arrow indicates from to Determining when to use them is based on whether the short is sufficient to visualize it LO9 Now an update… Nomenclature is evolving and updated regularly as techniques continue to be utilized and new findings analyzed Complex oncology microarrays and comparative analyses are updated to reflect the updated information available through the human genome projects The latest information utilizes prenatal analyses on maternal serum to give a better idea of expected outcomes ISCN recommendations provide the foundation but Cytogenetics Nomenclature is not a static thing Nomenclature is an evolving part of the field of Genetics LO9 International System for Cytogenetic Nomenclature International language for describing genetic changes https://www.ncbi.nlm.nih.gov/nlmcatalog/101771277 A new edition was published in 2020 so we are due for another update in the next year or two Questions On which type of chromosome would you expect to find a nucleolar organizing region (NOR)? (LO4) What is considered the typical, healthy male karyotype if no chromosome abnormalities are observed (a.k.a. traditional/normal male karyotype)? (LO1 and LO8) What would you expect to see if (LO3): ◦ There was a mutation in condensin that prevented its incorporation on chromosome 3? ◦ prevented its breakdown? ◦ Histone variants typically associated with a centromere were incorporated into a region near the telomere for a metacentric chromosome? What would you expect to see if (LO6/LO7): ◦ Telomeres became critically short? ◦ Telomerase became inactive in adult stem cells?