Molecular Biology

Chapter 13 Outline

Chromatin structure:

∑ There are four conserved core histones (H2A, H2B, H3 and H4) and one poorly conserved histone (H1/H5).

∑ Nucleosomes are the first order of chromatin folding; Nucleosomes are comprised of a core histone octamers (two copies of each core histone protein) plus one copy of H1; Nuclease digestion of chromatin yields 200bp fragments corresponding to a nucleosomes; DNA wraps around the histone octamer (F13.3); A core particle consists of a histone octamer plus 146bp of DNA; Each histone consists of 3 alpha helices, two loops and an extended N-terminal tail (F13.4); DNA length is condensed 7-fold by nucleosomes (F13.5).

∑ Histone H1 is found at the entry/exit point of DNA (F13.6); Histone H1 leads to a zig-zag structure and can easily be removed in low salt (F13.7, F13.8).

∑ The second order of chromatin folding is 30nm fibers; 30nm fibers form at increased ionic strength (F13.10); One structure for 30nm fibers is a solenoid produced through interactions among H1 histones (F13.11); Other structures for 30nm fibers are possible; H1 to H1 interactions are involved in 30nm fiber formation; 30nm fibers condense chromatin ~ 7 fold.

∑ A third order of chromatin folding is the 50nm fiber; The 50nm fiber involves formation of loops (F13.13); Loops are held at their base to the chromosomes scaffold and supercoiled (F13.14); Each loop is from 35kb-85kb.

Effect of chromatin structure on gene activity:

∑ Activity of Xenopus oocyte and somatic 5S RNA genes; Correlation of transcription and loss of H1 (F13.16); Addition of one H1 to each 200bp of DNA leads to repression of oocyte 5S RNA genes (F13.17); 5S RNA gene activity is determined by a competition between TFIIIπs and histones (F13.18, F13.19).

∑ Addition of an equal mass of core histones to a PolII promoter results in ~75% reduction in transcription (F13.20); The remaining 25% of transcription in the presence of core histones is due to 25% of promoters not tied up in histones (F13.21; F13.22); Activators can not counteract the repression by core histones.

∑ H1 can lead to a drastic repression of transcription that can be counteracted by activators (F13.23, F13.24); GAGA binding factor is an antirepressor, but doesnπt stimulate transcription; Antirepression is caused by removing nucleosomes that obscure a promoter or prevents nucleosome binding to the promoter.

∑ The regulatory regions of actively transcribed genes are devoid of nucleosomes (F13.25); Locating nucleosome-free zones on the SV40 minichromosome (F13.26, F13.27); DNase hypersensitivity assays reveal nucleosome-free regions in promoters (F13.28 ≠ F13.30).

∑ Nucleosomes are found in specific positions within regulatory regions of active genes; Use of micrococcal nuclease to define nucleosome positions via in vivo footprinting (F13.31).

∑ Histone acetylation correlates with gene activation; Histones are acetylated on lysines in the N-terminal tail, thus neutralizing the positive charges and loosening their hold on DNA; Histone H3 and H4 are acetylated by two types of histone acetylases (HATs), HAT Aπs and HAT Bπs; HAT Bπs are found in the cytoplasm and acetylate H3 and H4 so they can be assembled into nucleosomes; HAT Aπs are found in the nucleus and acetylate histones that already have some acetyl groups; The bromo domain of HAT Bπs enable binding to acetylated H3 and H4; The coactivators Gcn5p, CBP and TAF_II250 are all HAT Aπs that could co-activate by opening up the promoter.

∑ Histone deacetylases repress transcription; Transcriptional repressors such as nuclear receptors not bound to hormone interact with co-repressors (Sin3 and NcoR/SMRT) and histone deacetylases (F13.34); Mad-Max dependent repression occurs through association of Mad with Sin3 and HDAC-2 (F13.35); Interactions among thyroid receptor dimer (TR-RXR) and co-repressors/deacetylases or coactivators/HAT Bπs determine the acetylation state of histones and gene activity (F13.36); Histone acetylation also weakens interactions between adjacent nucleosomes.

∑ The Swi/Snf and ISwi genes are coactivators that remodel chromatin by removing or moving nucleosomes from promoters.

∑ Heterochromatin is highly condensed DNA that is inaccessible; Genes within 3kb of a telomere are silenced due to telomeric heterochromatin; The RAP1 and SIR2, SIR3 and SIR4 proteins are involved in telomeric heterochromatin formation (F13.39); Histones in telomeric heterochromatin are hypoacetylated.

∑ Nucleosomes are displaced during transcription (F13.41, F13.42); Nucleosomes move outside the transcribed region when DNA is transcribed (F13.43, F13.44); Nucleosomes are generally displaced without dissociating from DNA (F13.45, F13.46).