Main Ideas - Chromatin= DNA+ proteins. It is highly structured. Loose DNA double helix -> metaphase chromosome= 10,000x packaging!- Structural unit of chromatin = nucleosome with 4 core histone proteins, H2A, H2B, H3, H4!- Histones can carry many diff. modifications that med. structure and condensation state of the chromatin!- 3 maintenance structures of chromatin are: replication origins (many), telomeres (2/chromosome), and centromeres (1/chromosome)!- Chromosomes are duplicated during S phase. Resulting 2 halves of each chromosome (sister chromatids) are sep. during mitosis.!- In a mitotic cell division, one cell splits into 2 daughter cells that are genetically identical to the mother cell and each other!!!Learning Objectives!1) Explain the structure and organization of chromatin!2) Explain how histone modifications influence chromatin organization!3) Describe the 3 maintenance structures of chromosomes !4) Describe the stages of chromosome segregation in mitosis!5) Relevance for med practice and disease!!1) Chromatin - DNA is packed into chromatin in euks!- DNA wrapped around histone 2x= nucleosome (146bp)-> this is beads on a string-> pack up nucleosomes to make 30 nm fiber= chromatin fiber-> attach this to protein scaffold-> condense into chromosome!- chromatin packing (S phase)= DNA compressed 40x!- metaphase chromosomes= DNA compressed by another 200x!- Structural unit= nucleosome!- Core= 4 histone pairs: H2A, H2B, H3, and H4 with H1 (linker histone) on the outside!- Nucleosomes separated by 50/60 DNA bp stretch!- Nucleosomes= dynamic structures, DNA unwraps/rewraps on one about 4 times/sec. to give proteins access to the DNA (ex. transcription factors, DNA polys, DNA repair...)!- Chromatin remodeling factors target nucleosomes to catalyze nucleosome sliding and making the DNA available/open. They are ATP-dep. protein complexes. !- Nuclear scaffold/matrix proteins help organize the chromatin. Scaffold is complicated and has lots of proteins. !- S phase- it has RNA, ribonucleoproteins, DNA/RNA polys, histone acetyl transferase, and topo II!- M phase- it is a condensin complex of SMC proteins (structural maintence of chromosomes). This helps to hold the sister chromatids together during mitosis. !!2) Histone modifications-> chromatin organization - Histone mods (mod. at tail, tail is sticking out away from the histone core)!- many possible, gives cells flexibility !1) Histone acetylation--done at lysine residues by HATS (Histone acetyl transferases) to weaken DNA/histone interactions-> by neutralizing the charge of the histone-> supports transcription (proteins can get at the DNA). Counteract by HDAC (deacetyl)!2) Histone methylation--at lysine or arginine, has opp. effects on packaging dep. on residue position!3) Histone phosphorylation--on serines, req. for condensation of chroms in mitosis/meiosis!- Histone variants!- Have special functions!- CENP-A= only at centromere for centromere function and kinetochore assembly!- H2AX= for DNA repair and recombination, DNA double strand break repair!- Histone Code= histone modifications + histone variants!- Not DNA seq. specific, complex, lots of possibilites!- Code-reader complex= reads histone code and mediates cell response!- DNA mods!- methylation of cytosines->chromatin condensation-> gene silencing= genomic imprinting basis! 3) Maintenance structures of chromosomes!- Replication origins: !- euks start chrom. replication at many sites (1/100,000 bp)!- starts with binding of pre-RC, chroms replicated once/cell cycle during S phase (syn. phase= interphase)!- Bidirectional movement along the chromosome. Leading strands go towards/into each fork.!- Telomeres:!- protect chromosome ends!- repetitive tandem sequences (TTAGGG). These telomeric repeats bind proteins that cap chrom. ends= shelterin complex!- telomerase added TTAGGG to the telomeric ssDNA ends to maintain telomere length!- our telomeres shorten with age!- most somatic cells don't have telomerase!- Centromeres!- region of the DNA where the sister chromatids and joined together!- has specific DNA seq. usually tandem repeats= satellite DNA - Has specific histone H3 variant= CENP-A-> helps to assemble the kinetochore!- MTs assemble during mitosis to pull the sister chromatids apart!- Phases of cell cycle!- G1= growth face, DNA replication factors synthesized, cellular contents duplicated!- S= chromosomes duplicate. each chrom. turns into 2 sister chromatids!- Can see euchromatin (light area) and heterochromatin (dark area) in an S phase nucleus!- Euchromatin: lightly packed, gene rich, actively transcribed, methylated lysine 4 on histone H3 - Heterochromatin: tightly packed, gene poor, not transcribed, tight nucleosome assembly, methylated lysines 9/27 on histone H3!- G2= cell double checks, repairs. DNA amount is double but ploidy (chromosome #) is the same!- Mitosis!- Cytokinesis!!4) Mitosis - Prophase: centrosomes move to opp. poles, spindle MTs form, chroms condense!!!!!!!!!!!- Prometaphase: nuclear envelope breakdown, chroms attach to spindle MTs via kinetochores!- Metaphase: chromosomes align at the equator of the spindle. Kinetochore MTs attach sister chromatids to opp. poles (don't care about homolog. chroms)!- Anaphase: sister chromatids separate-> daughter chromosomes. Chroms pulled apart by MT shortening and spindle poles moving apart!- Telophase: daughter chroms arrive at opp. poles, nuclear envelope reassembles!...Cytokinesis: cytoplasm divided in 2 via contractile ring of actin + myosin!All chromosomes behave independently in mitosis! 2 homologs shouldn't interact. 5) Med. Relevance!- Drugs that target chromatin remodeling!- HDAC inhibitors (HDI)-> hyperacetylation-> increased gene transcription!- HDI-> psychiatry, neurology-> mood stabilizers, anti-epileptics, cancer therapy (Vorinostat, Romidepsin)!- Telomere diseases!- Telomere shortening: aging disorders, alz, werner syndrome, parkinsons, bone marrow failure!- Unwanted maintenance of telomere length: cancer, mediates cell immortality!- Telomerase inhibitors for cancer treatment currently being developed!- Mitotic spindle inhibitors for chemotherapy!- ex. Paclitaxeel (Taxol): treat tumors!- Many centrosome-assoc. diseases: cancer, infertility!- Loss of Heterozygosity during mitosis!- Missegregation of chromosomes: when sister chromatids don't separate and are pulled to one pole together (one cell has trisomy, one has monosomy for that chrom.). Cancer!- Mitotic recombination: between a chrom.
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