Controlling Chromatin Structure

Model ? Transcription in vitro in vivo DNA TFs RNA polymerase nucleosomes Model ? 1-Equilibrium model: prokaryotes (evidence for semi-conservative. replication of the DNA molecule) 2-Discontinuous change-of-state: eukaryotes EXAM

DNA binding depends on protein concentration Protein factors What is (are) the limiting factor(s)? Example: Transcription in bacteria

Chromatin can have alternative states Inactive--DNA/histones Active--DNA/RNApolymerase/TFs Figure 30.2 RNA polymarase/TFs NO TRANSCRIPTION HISTONES TRANSCRIPTION “The addition of either TFs or nucleosomes may form stable structures that can not be changed by modifying the equilibrium with free components” How is the chromatin structure regulated?

Chromatin Can Have Alternative States Figure 30.2 Chromatin structure: is stable cannot be changed by altering the equilibrium of transcription factors and histones

Chromatin Remodeling Is an Active Process Remodeling complex (protein) There are several chromatin remodeling complexes that use energy provided by hydrolysis of ATP (ENERGY) Release of nucleosomes Binding of TFs and RNA poly Figure 30.3

Chromatin remodeling is an active process Expose DNA Nucleosome Sequence Spacing-out Release

Nucleosome Organization May Be Changed at the Promoter Activator Remodeling complexes are recruited to promoters by sequence-specific activators. The factor may be released…. once the remodeling complex has bound. Remodeling complexes are proteins that do not recognize specific DNA sequence. All are ATPases complexes Mechanisms--not well know-- --the ATP hydrolysis is used to twist the DNA by creating a mechanical forces-- Figure 30.6

The SWI/SNF, RSC, and NURF complexes all: are very large they share some common subunits A remodeling complex does not itself have specificity for any particular target site. It must be recruited by a component of the transcription apparatus. CTD tail of the RNA pol II Figure 30.5

Histone modification is a key event Silencing--gene -local effect --heterochromatin-regional effect How? Binding of additional proteins Modifications--methylation (Met) --acetylation (Ace) --phosphorylation (Pho) Effect? Reduce positive charge Consequence? Ace--Active Met-Inactive

Histone Modification Is a Key Event Histones are modified by: methylation acetylation phosphorylation Figure 30.8

Histone Acetylation Occurs in Two Flavors Histone acetylation occurs transiently at replication and during gene activation. During DNA replication Before Histones are Acetylated before the incorporation into the nucleosome. Function--requires for the assembly/ structure of new nucleosomes --recognizes new factors (Enzymes: Histone Acetyltransferases-HAT) Histones are Deacetylated after the incorporation into the nucleosome. Function-- required for gene activaton (Enzymes: Histone Deacetylases-HDAC) Figure 30.12

Histone acetylation is associated with activation of gene expression. During gene activation Histones are Acetylated after the incorporation into the nucleosome. Function--recognizes new factors (Enzymes: Histone Acetyltransferases-HAT) Figure 30.13

Acetylases Are Associated with Activators Deacetylated chromatin may have a more condensed structure. Transcription activators are associated with histone acetylase activities in large complexes. Figure 30.14

Histone acetylases vary in their target specificity. Acetylation could affect transcription in a quantitative or qualitative way. HAT- Acetylase complex P300 targets H4 PCAF targets H3

Deacetylases Are Associated with Repressors Deacetylation is associated with repression of gene activity. Deacetylases are present in complexes with repressor activity. DHAC- Deacetylase complex Rpd3 targets H4/H3 Figure 30.16

Constitutive Facultative Eurochromatin vs Heterochromatin

Methylation of Histones and DNA Is Connected Methylation of both DNA and histones is a feature of inactive chromatin. The two types of methylation event may be connected. Histone methylation is associated with gene inactivation -H3 Lys 9, Lys 4- and condensation of chromatin- H4 Arg 3- DNA methylation is associated with gene repression CpG regions

Chromatin States Are Interconverted by Modification Acetylation of histones is associated with gene activation. Methylation of DNA and of histones is associated with heterochromatin. Figure 30.17

Promoter Activation Involves an Ordered Series of Events Key element The remodeling complex may recruit the acetylating complex. Acetylation of histones may be the event that maintains the complex in the activated state. Promoter activation involves an ordered series of events Activation Figure 30.18

Glucose Regulation of β-pancreatic Cells (Blood) Sugars Blood

Histone Phosphorylation Affects Chromatin Structure At least two histones are targets for phosphorylation, possibly with opposing effects. What kind of phosphorylation? Serine Threonine Tyrosine H1 Ser phosphorylation during mitosis by cd2 kinase (control chromatin condensation)

Some Common Motifs Are Found in Proteins That Modify Chromatin The chromo domain is found in several chromatin proteins that have either activating or repressing effects on gene expression (methylated sites). The SET domain is part of the catalytic site of protein methyltransferases. The bromo domain: is found in a variety of proteins that interact with chromatin is used to recognize acetylated sites on histones.

What is mechanism that control chromatin structure? Genes Su(var) and E(var) (60 genes) Su (suppressors) and E (enhancers) factors are enzymes that respond to environmental and metabolic signals. HP1(Heterochromatin protein 1) --structure:chromo domain --bind metylated H3 lys position 9 SUV39H1 -SET domain- methyltransferase Variegation 9 14 (Ace)

Some Common Motifs Are Found in Proteins That Modify Chromatin The chromo domain is found in several chromatin proteins that have either activating or repressing effects on gene expression (methylated sites). The SET domain is part of the catalytic site of protein methyltransferases. The bromo domain: is found in a variety of proteins that interact with chromatin is used to recognize acetylated sites on histones.

Epigenetic Effects...

Epigenetic effects can result from: 31.1 Introduction Epigenetic effects can result from: modification of a nucleic acid after it has been synthesized by the perpetuation of proteins Epigenetic: Functional Genomic Two Methylation===gene inactivation Figure 31.1

HP-1 How do heterochromatin proteins propagate? Figure 31.02: Self-assembling (self-templating) complexes maintain heterochromatin. HP-1 How do heterochromatin proteins propagate? H3 and H4

Heterochromatin is nucleated at specific DNA seq. and propagates. Figure 31.3 Heterochromatin is nucleated at specific DNA seq. and propagates. The length of the inactive region varies from cell to cell: Inactivation of genes in this vicinity causes position effect variegation. Similar spreading effects occur at: Telomeres (telomeric silencing) the silent cassettes in yeast mating type locus ( MAT, HML and HMR) Functional DNA effect

What is mechanism that control chromatin structure? Genes Su(var) and E(var) (60 genes) Su (suppressors) and E (enhancers) factors are enzymes that respond to environmental and metabolic signals. HP1(Heterochromatin protein 1) --structure:chromo domain --bind metylated H3 lys position 9 SUV39H1 -SET domain- methyltransferase Variegation 9 14 (Ace)

31.3 Heterochromatin Propagates from a Nucleation Event Heterochromatin is nucleated at a specific sequence. The inactive structure propagates along the chromatin fiber. Genes within regions of heterochromatin are inactivated. Nucleation (DNA-Methylation) (Histone-Methylation) Propagation (Self- aggregation) Figure 31.4

31.3 Heterochromatin Depends on Interactions with Histones HP1: is the key protein in forming mammalian heterochromatin acts by binding to methylated histone H3 Figure 31.5 Nucleation Propagation

The targets of Rap1 include: Rap1 initiates formation of heterochromatin in yeast by binding to specific target sequences in DNA. The targets of Rap1 include: telomeric C-A repeats silencers at HML and HMR Rap1 recruits Sir3/Sir4. These interact with the N-terminal tails of H3 and H4. Figure 31.8 (C-A) SIR1,2,3,4 SIR1 ( seq. specific binding ) SIR2 (deacetylase) Silence information regulator (SIR)

31.6 Chromosome Condensation Is Caused by Condensins SMC (structural maintenance chromosome) proteins are ATPases that include condensins cohesins A heterodimer of SMC proteins associates with other subunits. (condensation of Chromosome) (linking both chromatids) Figure 31.13

V-shape structure --Coiled-coil structure --ATPase and DNA binding (N- and C-terminal regions) Cross-links the DNA Dimerization

Condensins are responsible for condensing chromosomes at mitosis. The condensins cause chromatin to be more tightly coiled by introducing positive supercoils into DNA. Condensins are responsible for condensing chromosomes at mitosis. Chromosome-specific condensins are responsible for condensing inactive X chromosomes in C. elegans. Figure 31.16

Epigenetic effects can result from: modification of a nucleic acid after it has been synthesized by the perpetuation of proteins by protein aggregates (Prions-Mad Cow disease-Prp gene) DNA

--DNA transcription and repair Acetylation, Methylation, Phosphorylation, Ubiquitylation: --DNA transcription and repair --Proteosome (cell cycle) Sumoylation: Small Ubiquitin-like Modifier --Transcriptional regulators --Enzymatic activity E1