Controlling Chromatin Structure Why? It is related to gene expression and regulation

Model ? Transcription in vitro in vivo DNA TFs RNA polymerase nucleosomes Model ? 1-Equilibrium model: prokaryotes ( limited energy) (evidence for semi-conservative. replication of the DNA molecule) 2-Discontinuous change-of-state: eukaryotes ( non-limited energy) Dynamic, mutations and Cancer

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

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 -coding regions- What about non- coding regions? Figure 30.5

Mol Cell 2013 49(2): 298-309.

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)