Eukaryotic transcription (III)

Eukaryotic transcription (III)

1. Chromosome and chromatin structure Chromatin, chromatid, and chromosome chromatin Genomes exist as chromatins before or after cell division (interphase) but as chromatids or chromosome during cell division chromosomes

Euchromatin and heterochromatin Euchromatin: less condensed chromatin, light color under microscope, actively transcribed genomic regions Heterochromatin: highly condensed chromatin regions, such as centromeres and telomeres, which are not actively transcribed.

How chromatin structure affects transcription activity of genes? The degree of chromatin condensation (i.e. how tightly is chromatin packed) affects transcription. Usually the less condensed euchromatins are active because they are transcribed more readily than the highly condensed heterochromatins ( silent ).

Nucleosomes are the basic structural units of chromatins 10 nm 35-85 kb per loop

Nucleosome and histones Nucleosomes are DNA-histone particles. Histones are basic proteins rich in Arg and Lys, they bind tight to negatively charged DNA. Nucleosome consists of 147 bp DNA wrapped around 8 core histone molecules. Core histones are H2A (~13 kd) H2B (~14 kd) H3 (~15 kd), H4 (~11 kd). Nucleosomes are linked by about 50bp linker DNA and linker histones H1 (~21kD). Histones are highly conserved.

Summary of chromatin structure Linker histone H1 (10 nm fiber) Nucleosomes composed of core histones 2x(H2A, H2B,H3, H4) 147bp DNA

2. Nucleosome and transcription 1). The position of nucleosomes is affected by the number of linker histone H1, histone modification, and chromosome remodeling proteins. 2). An active promoter is usually free of nucleosomes 3). Nucleosome localization is dynamic (nucleosome "moves"), the location and density of nucleosomes can be changed by the histone modification and chromatin remodeling process. 4). Histone modifying enzymes and chromatin remodeling proteins play critical roles in chromatin remodeling to affect transcription.

(1) Linker histone (H1) may inhibit transcription Nucleosomes are connected by histone H1, also called linker histone. H1 often represses transcription. H1 may repress transcription by simply occupying the position of DNA that is normally accessible to transcription activators. nucleosome H1

(2) Active promoter is free of nucleosomes SV40 minichromosome uncut BamHI or Bgl I uncut SV40 minichromosom e Experiment: 1. BglI cuts a circular SV40 viral minichromosome within the promoter and enhancer (P/E) region. 2. BamHI cuts at a site opposite to the P/E region. 3. The BamHI-cut SV40 DNA shows a nucleosome-free DNA (arrow) in the center, BglI-cut SV40 shows nucleosome-free DNA at the end. Conclusion: promoter/enhancer DNA (P/E) must be free of the nucleosome BamHI BglI

(3). Chromatin remodeling a). Chromatin remodeling (or chromosome remodeling) is an ATPdependent process that changes the chromatin architecture. Chromatin remodeling is accomplished by changes of the position and/or density of core nucleosomes. b). chromatin remodeling proteins or chromatin remodeler, such as SWI/SNF, SWR, ISWI, INO80, NuRD, etc consume ATP to change chromatin architecture. c). Chromatin remodeling can be accomplished by change of core histones or histone modifications. d). Chromatin remodeling may activate or repress transcription.

Mechanisms of chromatin remodeling Chromatin remodeling complexes (CRC) use hydrolyze ATP to change the nucleosomal position or density by at least 4 mechanisms. 1) Mobilization of nucleosome position (sliding), 2) Dissociation of DNA-histone contact (unwrapping), 3) Remove core histones (histone eviction). 4) Replacement of the common histone unit (exchange of histone variants), such as change from H2A to H2A.Z.

chromatin remodeling by replacing core histones (1) Nucleosomes are tightly packed by nucleosomes composed of the common H2A, H2B, H3, and H4. (2-3) A remodeler protein (pink) facilitates reposition the nucleosome to establish a nucleosome-free region (red). (4) Another remodeler protein (brown) facilitates replacement of the common core histone H2A (green) with the histone variant H2A.Z (dark pink) into nucleosomes flanking the nucleosome-free region. (5) The chromatin now possesses a stable nucleosome-free region that allows for productive transcription.

Histone modifications facilitates chromatin remodeling

2. Histone modifications (1) N-terminal of about 11-36 amino acids of core histone (H2A, H2B, H4, H4) are called histone tails. Histone tails are not buried inside a core nucleosome, and often chemically modified. (2) The same residue of a histone may be modified by different chemical reactions under different conditions. (3) The same chemical modification in different residues of a histone may have different effect on transcription. (4) The combined pattern of histone modifications is referred to as histone codes. e.g.h3r2mk4mk9ack14ac This code means a histone H3 with Arg2 methylated, Lys4 methylated, Lys9 acetylated, Lys 14 acetylated, (5) Histone code is important to transcription

Four major histone modifications Acetyl- Methyl- Phosphoryl- ubiquitin Acetylation: addition of acetyl group to lysine residues. Acetylated histones have less positive charges and interact poorly with DNA. Histones in euchromatin are highly acetylated (hyperacetylation), histones of heterochromatin are rarely acetylated (hypoacetylation) Methylation: it often occurs to the same Lys residue as acetylation, so histone methylation competes with acetylation. It also occurs to Arg. Phosphorylation: to introduce negative charges Ubiquitination: Although poly-ubiquitination is usually a degradation signal of the ubiquitinated proteins, mono-ubiquitination (add only one ubq) is not a degradation signal. Mono-ubiquitination is part of histone code.

(1) Histone acetylation and deacetylation HAT (Acetyl-CoA) HDAC 1. Histones can be acetylated. HAT (histone acetyltransferase) transfers acetyl groups from the donor acetyl-coa to the lysine residues of histones. Only lysine (but not arginine) of histones are acetylated. Acetylated histones can be deacetylated by HDAC (histone deacetylase) that removes the acetyl group from histones. 2. Lysine acetylation neutralizes positive charges of histone. Histone acetylation tend to reduce the interactions between the neighboring nucleosomes, decreasing the overall chromatin condensation. Therefore, histone acetylation usually (not always) activates transcription.

activator to repressor conversion Transcription co-activators often recruit HAT to activate transcription. Transcription corepressors often recruit HDAC to repress transcription. When Max binds Myc, the Max-Myc dimer recruits HAT (CBP and GCN5) to acetylate histones, which activates transcription. When Max binds to Mad, the Max-Mad dimer recruits HDAC to deacetylate histone, resulting in suppression of transcription.

Experiment 1. Transform cells with a plasmid expressing FLAG-tagged HDAC2, or FLAG only (control), with or without the second plasmid expressing Mad1 or a mutant Mad1 (Mad1Pro). V= cells transformed with vector only. 2. Immunoprecipitate (IP) with anti-flag antibody, run IP in SDS-PAGE 3. Immunoblot with anti-sin3 (=msin3a) antibody first, strip the membrane and re-probe with the anti-mad1 antibody Result: 1. IP from the control (FLAG-only) cells pull down nothing. 2. IP from FLAG- HDAC2 cells pull down Sin3 regardless of Mad1. 3. IP of FLAG- HDAC2 cell also pull down Mad1, but not mutant Mad1 (Mad1Pro).

It was previously known that: - Sin3 can interact with HDAC2 but not DNA, so it is a co-repressor - Mad1 is a transcription repressor interacting with DNA and Sin3, - mutant Mad1Pro does not interact with Sin3 or inhibit transcription. Question: How does Mad1 repress transcription? Reasoning: It was found by the co-ip experiment that: 1. FLAG alone did not pull down Mad1 or Sin3; 2. FLAG-HDAC2 pulled down Sin3 in all cells (as previously known); 3. FLAG-HDAC2 pulled down Mad1; 4. FLAG-HDAC2 did not pull down mutant Mad1Pro. Conclusion: Mad1 must interact with the Sin3-HDAC2 complex to repress transcription. When Max binds to Mad1, the Max-Mad1 dimer recruits HDAC2, via the Sin3 bridge. HDAC2 deacetylates histones around the promoter region, resulting in inhibition of transcription. Sin3 HDAC2

Repressor to activator conversion Without thyroid hormone (TH), the thyroid receptor (TR) binds co-repressor NcoR to recruit Sin3, which binds HDAC to deacetylate histone and repress transcription With thyroid (TH), TR binds to the coactivator CBP-CAF-TAF250, which is a HAT complex that acetylates histone to activate transcription

Using ChIP (chromatin-imunoprecipitation) to identify chromatin DNA that is bound by a protein in vivo 1. Isolate total chromatins, crosslink chromatins to fix DNA-protein interaction 2. Cut chromatins to smaller fragments (e.g. about 500bp) by ultrasonication or mild nuclease digestion 3. Immunoprecipitation using antibodies against the transcription factor or modified histones (such as acetylated histone). The chromatin fragments bound to the protein are precipitated. 4. PCR amplification of DNA of the promoter of interest. The DNA of chromatin fragments bound by the protein (recognized by the antibody) should produce a PCR product.

Histone acetylation occur before transcription starts Experiment: 1. Infect cells with virus. 2. Isolate chromatin at different time after infection. 3. ChIP by antibodies against TBP or acetylated histone (e.g. α-ach4 K5, K8 is the antibody against histone H4 acetylated at the Lys5 or Lys8). 4. PCR the promoter DNA of the IFN- gene (encoding an interferon involved in immune responses) to examine how TBP or modified histones bind to the IFN- promoter DNA. Results: Acetylation of histones on the IFN- promoter occurred after viral infection but before TBP binding to the promoter and IFN- mrna expression.

Histone modification may also affect DNA-TBP interaction HDAC This model shows that the transcription repressor Mad/Max recruits HDAC to deacetylate histone and suppress transcription. In contrast, TAF250 (HAT) acetylate histone to facilitate TBP-DNA interaction between TATA box and TBP that is associated with TAF250. The ultimate level of transcription is dependent on the balance between HDAC and HAT.

(2) Histone methylation Histone can be methylated at both lysine and arginine residues. Methyltransferases (HMT) transfer (one to three) methyl groups from the donor SAM (s-adenosylmethionine) to lysine or arginine residues of histone tails. Methylation does not reduce the positive charges of lysine (or arginine), so it does not reduces nucleosome condensation. Instead, histone methylation often causes chromatin condensation and transcription inactivation.

Histone methylation may cause chromatin condensation to repress transcription If H3K9 is methylated, it binds to the chromosome remodeling protein HP1. HP1 recruits HMT, HMT methylates H3K9 in the next nucleosome, HP1 binds methylated histone to spread histone methylation and chromatin condensation to form heterochromatin. Heterochromatin cause gene silencing unless the gene is insulated from heterochromatin spreading by insulators.

(3) histone codes are dynamic and interactive Modification of one histone may affect other histones, different histone modifications have different effects on transcription. For example, H3S10 phosphorylation activates H3K14 acetylation, which inhibits H3K9 methylation and activates transcription. But if H3K9 is methylated first, it inhibits H3S10 phosphorylation and H3K14 acetylation, resulting in transcription repression.

A comparison Prokaryote RNAP II Eukaryote

Prokaryotic transcription: One RNA pol, holoenzyme of RNA pol =,,,ω + sigma factor Closed complex = holoenzyme + promoter DNA Promoters often contain TATA box at -10 position DNA regulatory elements (e.g. CAP-binding sites, operators, etc) are usually close to or within the promoter or the transcribed region Regulated by a few transcription factors, also regulated by changing of sigma factors, or even RNA polymerase (phage only) No histone, no DNA methylation, no complex chromatin structures Eukaryotic transcription: Three RNA polymerases, RNA pol II transcribes mrnas Holoenzyme of RNA pol II: more subunits, also need TFII s Preinitiation complex = holoenzyme + TFIID + promoter DNA Promoter often contains TATA box at -25 position DNA regulatory elements (e.g. enhancers and silencers) may be close to, within, or far away from the promoter or the transcribed region Regulated by many transcription factors, co-activator, and corepressors, also regulated by nucleosome position/density, histone codes, and other aspects of the chromatin structure.