Architectural TFs

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Architectural TFs
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Overview
DNA-binding TFs General principles
Architectural factors
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Recognition of response elementsActivators vrs
Architectural TFs

• Ordinary activators with sequence specific DNA
  binding
• Key recruitment sites for assembly of
  transcription complexes
• Architectural transcription factors playing a
  more structural role in the assembly of
  transcription complexes

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Architectural TFs - brief history

• Transcription activation - focus on more and more
  dimentions
• 70-ties 1-Dimentional understanding
• RNAPII TFs binding specific cis-elements
  required for selective transcription
• TFs mediate regulatory response
• 80-ties 2-Dimentional understanding
• Promoters/enhancers clusters of cis-elements
• complex regulation - Several buttons have to be
  pushed simultaneouly
• Ptashnes simplification - mixed order OK
• 90-ties 3-Dimentional understanding
• Three-dimentional assembly of TFs required for
  correct biological response

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3D protein-promoter complexes- factors dedicated
architecture

• some factors has a pure architectural function
• designated architectural transcription factors
• They lack a transactivation domain (TAD)
• Do not function out of their natural context (in
  contrast to ordinary acitvators)
• Their function is to confer a specific 3D
  structure on DNA

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Classical HMG-proteins

• non-histone chromatin proteins - original
  defining criteria
• high mobility in PAGE
• soluble in 2-5 TCA
• small lt 30 kDa
• High content of charged amino acids
• abundant 1 per. 10-15 nucleosomes

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Classical HMG-proteins

• Three classes of HMG DNA-binding proteins
• HMG-box family
• Eks HMG 1 and HMG 2
• Bends DNA substantially
• Facilitators of nucleoprotein complexes
• HMG-AT-hook family
• Eks. HMGI(Y)
• Antagonizing intrinsic distortions in the
  conformation of AT-rich DNA
• HMG-nucleosome binding family
• Eks. HMG14 and 17
• Mediates moderate destabilization of chromatin
  higher-order structure
• Not present in yeast or fly

HMGB
HMGA
HMGN
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HMGB-proteins
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HMG1 and 2

• 3 structural domains
• A and B with high homology (80-90 aa)
• acidic C-terminal
• Interaction with DNA (and histones?)
• A and B DNA
• C-term histone H1 or unknown function

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-
-
-
A
B
N
C
Histon H1?
DNA
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HMG-boxes in architectural proteins

• One or two HMG-box domains

30 Asp/Glu
acidic basic
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First eukaryotic architectural TF LEF1
(Grosschedl 1992)

• LEF1 a cell type-specific TF
• LEF1 contains an HMG-related domain
• LEF1 a sequence-specific TF that binds CCTTTGAAG
• found in enhancer of TCR?
• LEF1 induces strong bending of DNA - about 130o
• Induced bending brings nearly TFs in contact

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LEF1 3D
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LEF1 3D
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A whole family of architectural TFs with
HMG-domains

• UBF has repeated HMG-homologous repeats
• 4-6 ex dimer 10 HMG-like domains
• activator of rRNA gener
• UBF-DNA complex ? scaffold for SL-1 recruitment
• Interaction with 180 bp that is packed into a
  distinct structure
• DNA-motif in a series of TFs
• HMG-box designate the DNA-sequence-motif
• HMG-domain designate the protein motif

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Two subclasses of HMG-domain proteins

• Proteins with multiple HMG-domains
• low sequence-specificity
• Ubiquitous - found in all cell types
• eks. HMG1, HMG2, ABF-2, UBF
• Proteins with single HMG-domain
• (moderate) sequence-specificity
• Cell type-specific
• eks. LEF-1, SRY, TCF-1, Sox, Mat-a1, Ste11, Rox1

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Characteristic DNA-binding

• binds minor groove
• induce bending of DNA
• has high affinity for non-canonical
  DNA-structures such as
• cruciform DNA
• 4-way junctions
• cisplatin ? kinked DNA

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NMR-structures

• Examples
• HMG1 B-domain
• LEF-1
• SRY
• Yeast Nhp6p
• Drosophila HMG-D
• Common 3 helix L-form
• heliks II and III form an angle of about 80o
• Conserved aromatic aa in kink
• Basic concave side interact with DNA

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Similar structures of HMG domains
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Minor groove binding, intercalation and bending

• Objective shorten the distance between
  cis-elements facilitating interaction between
  bound factors
• DNA lt500bp relatively stiff ? induced bending
  required
• Mechanism for induced bending of DNA
• Protein scaffold
• HMG B-domain L-shaped protein
• TBP sadle
• Minor groove binding
• DNA-binding face hydrophobic surface that
  conforms to a wide, shallow minor groove
• 4 residues inserted deep into the minor groove
• Full or partial intercalation (kile)

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Intercalation in protein-induced DNA-bending

• Partial intercalation in the DNA helix of a
  protein side chain introduces a kink in the DNA
  enhancing the bend
• Large hydrophobic residues (N-term helix I)
  partially intercalates between two base pairs
• The A-box HMG domain has only an Ala in the X
  position not large enough to intercalate,
• Intercalation linked to bending also seen in
  other factors
• Partial (TBP)
• Inserted side chain unstacks two basepairs
• side chain as stacking-partner
• Full (ETS1)
• side chain penetrates into the helix
• side chain (Trp) as new stacking-partner
• Result helix axis direction altered

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Two points of intercalation, X and Y
Basic tail Binds Major groove
X only
Y only
X and Y
X major kink and intercalation site, Ysecond
kink due to partial intercalation
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Cooperation with TFs

• A major role of non-seq.spec. architectural
  factors is to facilitate formation of complex
  nucleoprotein assemblies
• Need interaction with sequence specific TF to be
  directed to precise locations
• An introduced bend could facilitate binding of
  one factor, and this could subsequently assist a
  second factor
• The seq.spec. architectural factors is known to
  participate in the formation of complex
  nucleoprotein assemblies like enhanceosomes
• TCRa and Interferon b

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Are all TFs architectural?

• A large number of publications TFx bends DNA
• positive reports TFx bends DNA
• negative reports TFx does not bend DNA
• All TFs that bind on one side of DNA will induce
  bending due to one-sided neutralization of charge
• Degree of bending will depend on ionic condition
• Uncertain if biologically relevant
• The term Architectural TFs should be reserved
  for factors with a particularly developed bending
  mechanism

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The charge neutralization model
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2. subgruppe HMGA

• .. First described by Søren Laland, an almost
  forgotten discovery

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HMGA - proteins with AT-hook

• The mammalian HMGI/Y (HMGA) proteins participate
  in a wide variety of cellular processes
• including regulation of gene trx and induction of
  neoplastic transformation and promotion of
  metastatic progression.
• All members have multiple copies of a DNA-binding
  motif called the AT hook'
• that binds to the narrow minor groove of
  stretches of AT-rich sequence.
• The proteins have little secondary structure in
  solution but assume distinct conformations when
  bound to DNA or other proteins
• Their flexibility allows the HMGI/Y proteins to
  induce both structural changes in chromatin
  substrates and the formation of stereospecific
  complexes called enhanceosomes'. Reciprocal
  conformational changes occur in both the HMGI/Y
  proteins themselves and in their interacting
  substrates.

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Members

• 4 known members
• Alternatively splicing gives rise to two isoform
  proteins, HMGA1a (HMGI) and HMGA1b (HMGY). These
  two are identical in sequence except for a
  deletion of 11 residues between the the first and
  second AT hook in the latter. Alternative
  splicing also produces HMGA1c.
• The related HMGA2 (HMGI-C) protein is coded for
  by a separate gene.
• Conserved
• Homologues of the mammalian HMGA proteins have
  been found in yeast, insects, plants and birds,
  as well as in all mammalian species examined.

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HMGA - AT-hook binding to DNA

• Each HMGA protein possesses 3 similar, but
  independent, AT hooks
• which have an invariant peptide core motif of
  Arg-Gly-Arg-Pro (palindromic consensus PRGRP)
  flanked on either side by other conserved
  positively charged residues.
• The HMGA proteins bind, via the AT hooks, to the
  minor groove
• of stretches of AT-rich DNA but recognize
  substrate structure, rather than nucleotide
  sequence.

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HMGA proteins heavily modified

• The HMGA proteins are among the most highly
  phosphorylated proteins in the mammalian nucleus.
• Cell cycle-dependent phosphorylation pga cdc2
  activity in the G2/M phase of the cycle.
• Sites T53 and T78 situated at the N-terminal
  ends of the 2. and 3. AT-hook. Phosphorylation
  significantly reduces (gt20-fold) DNA binding.
• HMGA proteins are the downstream targets of a
  number of signal transduction pathways that lead
  to phosphorylation.
• HMGA proteins are also acetylated
• at Lys65 by CBP and at Lys71 by PCAF
• as well as methylated and poly-ADP ribosylated
• Hypothesis Modifications may alter DNA-binding
  specificity?

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Architectural effects

• Architectural effects
• Binding of full-length HMGA proteins can bend,
  straighten, unwind and induce loop formation in
  linear DNA molecules in vitro.
• Multiple contact points with DNA may alter
  conformation of DNA
• A single AT-hook preferentially binds to
  stretches of 4-6 bp of AT-rich sequence, and
  partially neutralizes the negatively charged
  backbone phosphates on only one face of the DNA
  helix.
• The number and spacing of AT-rich binding sites
  in DNA influences the conformation of bound DNA
  and the biological effects elicited.
• HMGA may also induce conformational change in
  proteins
• HMGA forms protein-protein interactions with
  other transcription factors, which alters the 3D
  structure of the factors resulting in enhanced
  DNA binding and transcriptional activation.

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Maniatis HMGI(Y) contributes to formation of
enhanceosomes

• virus-inducible enhancer in the IFN-? gene (human
  interferon ?)
• cis-elements for NF-kB, IRF-1, ATF-2-c-Jun
• Synthetic (multiple cis-elements) enhancer ?
  natural
• Too high basal transcription
• Less induction
• Responds to several stimuli, while natural
  enhancer only responds to virus
• Biological function depends of HMGI(Y) as
  architectural component
• HMG I(Y)
• First described by Lund and Laland
• binds AT-rich DNA in minor groove (AT-hook)

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Recentverision
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Other functions of HMGA proteins

• HMGA and cancer
• HMGI/Y proteins are also involved in a diverse
  range of other cellular processes including
  pathologic processes such as neoplastic
  transformation and metastatic progression.
• Chromosomal translocations in a long 3.intron
• Intron 3 of the HMGA2 genes is extremely long
  (gt25 kb in human and gt60 kb in mouse) and
  separates the three exons that contain the AT
  hook motifs from the remainds of the
  3-untranslated tail region of the gene.
• Translocation within the exceptionally long third
  intron are commonly observed in benign
  mesenchymal tumors.

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3. subgruppe HMGN
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HMGN proteins

• Three functional domains of the HMGN proteins
• a bipartite nuclear localization signal (NLS),
• a nucleosomal binding domain (NBD)
• and a chromatin-unfolding domain (CHUD). The CHUD
  domain has a net negative charge.
• Binding of HMGN proteins to nucleosomes decreases
  the compactness of chromatin, and facilitates trx

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HMGN architectural elements reducing compactness
of chromatin

• Model of the binding of HMGN proteins to
  chromatin
• HMGNs interact with both the DNA and the histone
  component of the nucleosome
• The CHUD domain interacts with the amino terminus
  of histone H3.
• May also affect H1 binding
• Incorporation of HMGN proteins into chromatin is
  believed to reduce the compactness of the
  chromatin fiber.