Transcript Architectural TFs

Architectural TFs
MBV4230
Overview
DNA-binding
TFs
General principles
Architectural factors
MBV4230
Recognition of response elements
Activators vrs Architectural TFs
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Ordinary activators with sequence specific DNA
binding
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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
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Transcription activation - focus
on more and more dimentions
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70-ties: 1-Dimentional understanding
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80-ties: 2-Dimentional understanding
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≥ RNAPII: TFs binding specific cis-elements
required for selective transcription
TFs mediate regulatory response
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
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Three-dimentional assembly of TFs required for
correct biological response
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3D protein-promoter complexes
- factors dedicated architecture
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some factors has a pure architectural
function
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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
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non-histone chromatin proteins - original
defining criteria
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high mobility in PAGE
soluble in 2-5% TCA
small < 30 kDa
High content of charged amino acids
abundant: 1 per. 10-15 nucleosomes
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Classical HMG-proteins
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Three classes of HMG DNA-binding proteins
HMGB
HMG-box family
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HMG-AT-hook family
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Eks: HMG 1 and HMG 2
Bends DNA substantially
Facilitators of nucleoprotein complexes
Eks.: HMGI(Y)
Antagonizing intrinsic distortions in the conformation of AT-rich DNA
HMG-nucleosome binding family
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HMGA
HMGN
Eks.: HMG14 and 17
Mediates moderate destabilization of chromatin higher-order structure
Not present in yeast or fly
HMGB-proteins
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HMG1 and 2
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3 structural domains
A and B with high homology (80-90 aa)
 acidic C-terminal
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Interaction with DNA (and histones?)
A and B ≈ DNA
 C-term ≈ histone H1 or unknown function
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N
++++
A
++++
B
DNA
----
C
Histon H1?
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HMG-boxes in architectural proteins
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One or two
HMG-box
domains
30 Asp/Glu
acidic
basic
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First eukaryotic architectural TF:
LEF1 (Grosschedl 1992)
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LEF1: a cell type-specific TF
LEF1 contains an HMG-related domain
LEF1: a sequence-specific TF that binds
CCTTTGAAG
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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
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UBF has repeated HMG-homologous repeats
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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:
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“HMG-box” designate the DNA-sequence-motif
“HMG-domain” designate the protein motif
MBV4230
Two subclasses of HMG-domain
proteins
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Proteins with multiple HMG-domains
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low sequence-specificity
Ubiquitous - found in all cell types
eks.: HMG1, HMG2, ABF-2, UBF
Proteins with single HMG-domain
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(moderate) sequence-specificity
Cell type-specific
eks.: LEF-1, SRY, TCF-1, Sox, Mat-1, Ste11, Rox1
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Characteristic DNA-binding
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binds minor groove
induce bending of DNA
has high affinity for noncanonical DNA-structures
such as :
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cruciform DNA
4-way junctions
cisplatin  kinked DNA
+
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NMR-structures
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Examples
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HMG1 B-domain
LEF-1
SRY
Yeast Nhp6p
Drosophila HMG-D
Common: 3 helix L-form
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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
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Objective: shorten the distance
between cis-elements facilitating
interaction between bound factors
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DNA <500bp 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
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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,
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Intercalation linked to bending also seen in other
factors
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Partial (TBP)
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Full (ETS1)
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Inserted side chain unstacks two basepairs
side chain as stacking-partner
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
X and Y
Y only
X = major kink and intercalation site, Y=second kink due to partial intercalation
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Cooperation with TFs
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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
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The seq.spec. architectural factors is
known to participate in the formation
of complex nucleoprotein assemblies
like enhanceosomes
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TCR and Interferon b
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Are all TFs architectural?
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A large number of publications “TFx bends DNA”
positive reports “TFx bends DNA”
 negative reports “TFx does not bend DNA”
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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
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The term “Architectural TFs“ should be reserved for
factors with a particularly developed bending
mechanism
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The charge neutralization model
- - - - - - - - - - -- -- - - + - - - ++
--+
+
+
-+
+
+
+
--+
+
2
--3
+
-1
- - --Sp1
Asymmetrical charge neutralization
Bending
+ - -+-+- - - -+ +
+
- - - - -+-+
+ + - - -- +
- -- -+
+ +- - - - - ++- -+ + + ++ - - - +
-+ +
--- + + 2
--+
3
1
Sp1
Bending effect of charge neutralization
reduced in the pr esence of multvalent cations
2. subgruppe: HMGA
.. First described by Søren Laland,
an almost forgotten discovery
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HMGA - proteins with AT-hook
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The mammalian HMGI/Y (HMGA) proteins participate
in a wide variety of cellular processes
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All members have multiple copies of a DNA-binding
motif called the `AT hook'
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including regulation of gene trx and induction of neoplastic transformation and
promotion of metastatic progression.
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
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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
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4 known members
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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
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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
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Each HMGA protein
possesses 3 similar, but
independent, AT hooks
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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
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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 (>20-fold) DNA binding.
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HMGA proteins are the downstream targets of a number of
signal transduction pathways that lead to phosphorylation.
HMGA proteins are also acetylated
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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
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Architectural effects
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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.
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HMGA may also induce conformational change in
proteins
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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
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virus-inducible enhancer in the
IFN-b gene (human interferon b)
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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
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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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Recent
verision
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Other functions of HMGA proteins
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HMGA and cancer
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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 (>25 kb in human and >60 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
MBV4230
HMGN proteins
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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.
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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
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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

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Incorporation of HMGN
proteins into chromatin
is believed to reduce
the compactness of the
chromatin fiber.