Papers

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Papers

• Current Opinion Structural Biology10117 (2000)
• PNAS 971410914114 (2000)
• FEBS Letters 440 (1998) 264267
• Science 292 JUNE 2001
• Science 8 JUNE 2001
• J. Mol. Biol. (2001) 310, 1-26
• Proc. Natl. Acad. Sci. USA. 94, pp. 1522,
  January 1997
• Cell, Vol. 98, 811824, September 17, 1999
• Cell, Vol. 88, 741744, March 21, 1997

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Multiple binding sites affect transcription
McKnight and Kingsbury, 1982
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McKnight and Kingsbury
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McK K, cont.
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Multiple Elements Control Eukaryotic Transcription
6
Typical cis-acting elements
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Enhancers act at a distance
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Steric hindrance determines optimal spacing
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GAL4 Activator

• Acidic activation domain and DNA-binding domain

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Modular nature of Gal4 activator
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Other Activators
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GAL4 Chimera

• B) Chimeric protein with lexA DNA-binding domain
  wont bind to GAL4 site

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GAL4/LexA Chimera Binding
C) Chimeric protein binds at lexA site,
stimulates transcription
15
3 general types of activator domains

• Acidic
• Amphipathic helix, acidic amino acids on one face
• No consistent secondary or tertiary structure has
  been identified
• Glutamine-rich (Q-rich)
• Pro-rich (P-rich)

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No correspondence between type of DBD and type of
AD

• Examples of proteins with acidic AD
• GAL4 (Zn2Cys6)
• AP1 (bZIP)
• VP16 (no DBD)
• l repressor (HTH)
• Examples of proteins with Q-rich AD
• Sp1 (Zn finger)
• Antp (homeodomain)
• Oct (POU-homeo)

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Lack of fixed structure in activator domains

• DBDs of transcription factors form discrete
  structures that can be analyzed by X-ray
  crystallography and NMR
• The ADs do not generate identifiable electron
  density in the crystallographic analysis.
• This indicates that they do not form discrete
  structures.
• One hypothesis is that the ADs are unstructured
  until they interact with their targets.
• This is an induced fit model.

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Two-hybrid system
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more
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DNA-binding domains

• Homeodomain (homeotic genes and HOX)
• Zinc finger
• C4 (Steroid and other nuclear receptors)
• C2H2
• C6 (Gal4, fungal only)
• Forkhead (winged helix)
• Leucine zipper
• Helix-loop-helix

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Homeodomain
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Activators recruit RNA polymerase complex

• Some activators assist the binding of the
  transcriptional machinery (either a general
  transcription factor or RNA polymerase)

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Repressors prevent transcriptional activation

• Some repressors prevent binding of activators or
  general transcription factors
• Some repressors inhibit activity of activators or
  general transcription factors

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Variety of Ways to Control Regulatory Protein
Activity
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Steroid Hormones
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Steroid Receptors
Cortisol typical of steroid receptors, the
hormone displaces an inhibitory protein in this
case Hsp90
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Organization of Some Steroid Receptors
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Steroid response
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Variety of Ways to Control Regulatory Protein
Activity
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Activation by Protein Phosphorylation

• Gamma-interferon (IFNg) - hormone released by
  T-lymphocytes on antigen binding
• Binds to a membrane receptor
• Activates JAK kinase
• Phosphorylates Stata, which dimerizes
• Enters nucleus, binds response element,
  stimulating transcription of virus-response genes

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Nuclear receptors recruit chromatin-remodeling
enzymes through their AF2 motifs
HAT
CoA
ligand
- ligand
HAT
CoA
Gene X
Gene X
NRE
NRE
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Chromatin Structure

• DNA is supercoiled in the nucleus
• Association with histones produces nucleosomes

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Beads on a String and Solenoid
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Principal proteins in chromatin are histones
H3 and H4 Arg rich, mostly conserved
sequence H2A and H2B Slightly Lys rich, fairly
conserved H1 very Lys rich, most variable in
sequence between species
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Histone structure and function
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Histone interactions via the histone fold
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Nucleosome
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Nucleosome includes 8 histones
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Nucleosomes are the subunits of the chromatin
fiber

• Experimental evidence
• Beads on a string in EM
• Micrococcal nuclease digestion

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Solenoid
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Chromatin Packaging
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Chromosome localization in interphase
In interphase, chromosomes appear to be localized
to a sub-region of the nucleus.
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Heterochromatin
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Telomere Silencing
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Gene activation and location in the nucleus

• Condensed chromatin tends to localize close to
  the centromeres
• Pericentromeric heterochromatin
• Movement of genes during activation and silencing
• High resolution in situ hybridization
• Active genes found away from pericentromeric
  heterochromatin
• Silenced genes found associated with
  pericentromeric heterochromatin

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Nucleosome remodeling
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Chromatin remodeling ATPases are large complexes
of multiple proteins

• Yeast SWI/SNF
• 10 proteins
• Needed for expression of genes involved in
  mating-type switching and sucrose metabolism
  (sucrose non-fermenting).
• Some suppressors of swi or snf mutants are
  mutations in genes encoding histones.
• SWI/SNF complex interacts with chromatin to
  activate a subset of yeast genes.
• Is an ATPase
• Mammalian homologs hSWI/SNF
• ATPase is BRG1, related to Drosophila Brahma
• Other remodeling ATPase have been discovered.

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Chromatin remodeling ATPases catalyze stable
alteration of the nucleosome
II form a stably remodeled dimer, altered DNAse
digestion pattern III transfer a histone octamer
to a different DNA fragment
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Chromatin structure in active genes
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DNAse Protection
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Chromatin and transcription factors
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Transcription factor binding to DNA is inhibited
within nucleosomes

• Affinity of transcription factor for its binding
  site on DNA is decreased when the DNA is
  reconstituted into nucleosomes
• Extent of inhibition is dependent on
• Location of the binding site within the
  nucleosome.
• binding sites at the edge are more accessible
  than the center
• The type of DNA binding domain.
• Zn fingers bind more easily than bHLH domains.

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How are genes activated within chromatin?
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Some co-activators work on chromatin

• Transcriptional activation in vitro from some
  promoters requires a chromatin template
• Some co-activators and co-repressors covalently
  modify histones and transctipion factors
• Acetyl transferases
• Deacetylases
• Kinases, Methylases, ADP-ribosyltransferases
• Some co-activators use ATP hydrolysis to modify
  nucleosomes
• SWI/SNF, ISWI, etc

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Similarity between TAFs and histones
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Activating gene expression by covalent
modification of histones in chromatin
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Histones are acetylated and deacetylated
Histone acetyl transferases
Histone deacetylases
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Covalent modification of histone tails
N-ARTKQTARKSTGGKAPRKQLATKAARKSAP...- H3
4
9 10
14
23
27 28
18
N-SGRGKGGKGLGKGGAKRHRKVLRDNIQGIT...- H4
5
8
12
16
20
1
acetylation
phosphorylation
methylation
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Nucleosome
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Enzymatic modification of histones in chromatin
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Activators histone acetylation

• Some activators recruit histone acetylase, which
  adds acetyl groups to histones
• GCN4 binds to UAS, forms a complex including
  Gcn5, which acetylates histones and allows
  transcriptional machinery access to less
  condensed template DNA

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Nuclear HAT As are coactivators

• Gcn5p is a transcriptional activator of many
  genes in yeast. It is also a HAT.
• PCAF (P300/CBP associated factor) is a HAT and is
  homologous to yeast Gcn5p.
• P300 and CBP are similar proteins that interact
  with many transcription factors (e.g. CREB, AP1
  and MyoD).
• P300/CBP are needed for activation by these
  factors, and thus are considered coactivators.
• P300/CBP has intrinsic HAT activity as well as
  binding to the HAT PCAF.

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Yeast SAGA interacting with chromatin
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Repressors histone deacetylation

• Some repressors recruit histone deacetylase,
  which removes acetyl groups from histones
• In yeast, repressor Ume6 binds to URS1. A
  protein complex forms there, including Rpd3, a
  histone deacetylase.
• Deacetylated histones bind tightly, preventing
  binding of general transcription factors.

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Nuclear receptors recruit chromatin-remodeling
enzymes through their AF2 motifs
HAT
CoA
ligand
- ligand
HAT
CoA
Gene X
Gene X
NRE
NRE
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The yeast HO endonuclease gene requires both
SWI/SNF and SAGA

• The order of recruitment at the promoter
• 1. SWI5 activator sequence recognition
• 2. SWI/SNF complex remodel nucleosomes
• 3. SAGA acetylate histones
• 4. SBF activator (still at specific sequences)
• 5. general transcription factors
• Cosma, Tanaka and Nasmyth (1999) Cell 97299-311.
• The order is likely to differ at different genes

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A scenario for transitions from silenced to open
to actively transcribed chromatin
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From silenced to open chromatin
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Movement from hetero- to euchromatin
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Nucleosome remodelers and HATs further open
chromatin
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Assembly of preinitiation complex on open
chromatin
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Activator-dependent transcription from chromatin
in vitro involving targeted histone acetylation

• Kundu et al (Roeder lab)
• Mol. Cell 6551, 2000

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Panel A Artificial template, composed of gal4
sites, core promoter from Adenovirus major late
transcript gene, and template without G bases.
PanelB core histones purified from HeLa nuclei,
recombinant NAP1 (core histone chaperone) and
topoisomerase1 enzymes are used to reconstitute
the nucleosomal array on the template shown in
panel A.
D, another assay, limited micrococcal nuclease
digestion of the nucleosomal template to show
that there is physiological spacing between
nucleosomes. E, purified recombinant p300 used in
this study
C, supercoiling assay to show that nucleosomes
are being assembled onto the template.
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A, scheme of the transcriptional assay using the
assembled chromatin template.
B, transcriptional assay, using HeLa nuclear
extract (not defined system, need to be careful
when interpreting results). Assay show that p300
stimulates transcription from chromatin template
in activator and acetylation dependent manner.
But the truncated p300 HAT can not stimulate
transcription.
C, p300 only affects the activated transcription
on chromatin template in acetylation dependent
manner.
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Naked DNA
Chromatin
All three require p300 for activation of
chromatin template, although the extent of p300
mediated activation varied and correlated with
the strength of each activator.
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Conclusions

• p300 promotes transcription activation from
  chromatin by an activator, gal4-VP16.
• This p300 dependent transcriptional activation
  requires activator targeted acetylation of
  histones near the promoter.
• One control - show that gal4 DBD alone does not
  affect p300 dependent transcription.