long-range allosteric effect in gene transcriptional regulation

Ming LiGraduate School, CAS

Zhong-can Ou-yangInstitute of Theoretical Physics, CAS

International Symposium on the Recent Progress in Quantitative and Systems Biology , Dec 9-11, The Chinese University of Hong Kong

Outline

Stressed state of DNA in vivo

Model of topologically constrained DNA and duplex separation

Long-range Allosteric Effect and Database investigation: a case study

Part I

Stressed state of DNA in vivo

4 types of nucleotides: Adenine, ThymineGuanine, Cytosine

Waston-Crick base pair:A-T, G-C

Intrinsic right-handed helix, stiff polymer

B-DNA: uniform, sequence-independent

DNA: ~ centimeters Nucleus: ~ microns compaction ratio: ~1/8000

Twisting dsDNA is a highly efficient way for compaction !

Is the elastic response vital for DNA functioning ?

DNA Mechanics Plays a Role ?

eukaryote

prokaryote

Hierarchical architecture

DNA Stressed

S/MARs:Boundaries of topologically-independe

nt domains ?

bases of chromatin loops S/MAR

(Scaffold/Matrix Attachment Region)

Chromosome AssemblyChromatin Loop Model

DNA segment per nucleosome: ~167 bp

The segment is actually undertwisted :

one helical turn unwound per nucleosome.

torsional stress generated

Genomic DNA is potentially under unwinding stress

Sequence Heterogeneity Sequence Heterogeneity ? ? Structure Heterogeneity Structure Heterogeneity

bubble

cruciform

Chirality Variable Unstacking

Unwinding can promote local strand-separation (bubble) as well as global axis-supercoil

ing

Standard B-DNA

Local bubble

Unwinding stress in vivo Plays the Key Rolein Bubble formation !

In summary:

Part II

Benham Model of topologically constrained DNA &&sequence-dependent duplex separation

Lk : linking number, number of helical turns Lk0 : ‘linking number’ of relaxed DNA (uniform B-DNA) Lk0= N/10.5

σ : superhelical density. (Lk – Lk0)/ Lk0 σ< 0, negative supercoiling σ> 0, positive supercoiling

For eukaryotes, DNA is always unwound to a degree σ~ - 0.06 (1/167)

Characterizing the degree of unwinding …

Supercoiling free energy ( when σ << 1 )

2

22

1

qRTNG

21( )

2G K Lk

-0.06

10.5

92002200 q

Circular DNA Fully-stretched linearDNA with fixed ends

: bp number per helical turn of B-DNA

q is determined by the bending and twisting stiffness of dsDNA, as well as the topological constraints imposed on dsDNA

For more details on related DNA mechanics, see Ming Li, AAPPS Bulletin, Vol.16, No.3, 34-39

Bubble Formation is Sequence Dependent !

2N configurations{…10111111100…}

local bubble

a : initiation energy of bubble formation

jn = 0 … base paried

jn = 1 … base unparied

j : rewinding angle of the denatured region

ATb GCb : base unparing energy

When there are small fraction of unwound region, the supercoiling energy form slightly changes as:

N

jjnn

1

A

nTw total change in twisting turns

upon bubble formation

N

j

jjn

1 2

21 2

1( )

2

qRT nH

A

Bauer WR, Benham CJ., J Mol Biol. 1993, 234(4):1184-96.

Benham Model

total energy

Topological constraint long-range coupling between any two sites ! twisting energy of the two strands in bubble regions

unpairing energy in bubble (sequence dependent ) nucleation (initiation) energy for bubble formation (there can be multiple bubbles on a single DNA)

4321 HHHHH

21 2

1( )

2

qRT nH

A

j

jjnc

H 22 2

j

jjbnH 3

j

jj nnaarH )1( 14

8.10a 1molkcal

58.3c 21 radmolkcal

255.0ATb 1molkcal

301.1GCb 1molkcal

Parameter (illustrative) values under physiological condition

(the qualitative results shown later are actually quite insensitive to those values )

Unwounding Probability Profile

{ }

{ }

( 1)Hj

sj H

s

e n

pe

for any DNA sequence

Pi and Pj are tightly correlated due to the global topological constraints ( H1 !) imposed on the dsDNA, i.e., bubbles can be competitive in releasing the imposed twisting stress.

Can there be anything new (besides the individual denaturing events) introduced by topological constraints ? Long-range Allosteric Effect : the tele-communication between site A and B(protein binding onto the denatured site A may re-close the bubble and induce a new bubble at site B)

A

B

(DNA)

(protein)

B

A

It’s difficult to detect such a phenomenon in vivo by experiments, and it’s also almost impossible to directly ‘calculate’ such an effect quantitatively for real cases by taking account of every molecular detail (one can be drown in the details).

Beginning with everything , ending with nothing !

Anyway, How can one do anything meaningful ? --- Bioinformatics to rescue !

Bioinformatics offers an alternative approach:exploring the biological data to find the statistically significant patterns which may cast some light on the understanding of the underlying molecular mechanism

Part III

Long-range Allosteric Effect and Database investigation: a (bioinformatic) case study on SMAR function

D. menalongaster Histone gene cluster

5- —H3—H4—H2A—H2B—H1— -3MAR MAR

H3

H4

H2A

H2B

H1

SMAR SMAR

Convergently transcribed gene pairs: H4/H2A, H2B/H1

Coordinate transcription

relation between these two aspects?

S/MAR is detected between H1 and H3 by biochemical experiments

(S/MARt-DB: SM0000037 )

Some SMARs are observed as stress-induced unwound elements ( necessary but insufficient for chromatin loop formation? )

Bubble position coincides with annotated SMAR location

http://smartdb.bioinf.med.uni-goettingen.de/

SMARtDB/browse/index.html

SMAR binding to matrix: recovering the supercoiling stress on the intervening dsDNA

New unwound regions

downstream to convergently- transcribed gene pairs

relation to gene transcription , or even to the coordinate transcription of the whole gene cluster ?

5—H3—H4—H2A—H2B—H1—3

Competition Between Bubbles : Long Range Allosteric Effect (LRAE)

Adapted from: Wang, J.C. 1991. DNA topoisomerases: why so many? Journal of Biological Chemistry 266:6659-6662.

Twin-model of supercoiling domain in gene transcription

SMAR: retaining the negative supercoils around the unwound regions?? Buffer of the generated positive supercoils ??

Homologous gene locus:D.hydei Histone gene cluster

No record in SMARt DB; a prediction

Further support is given when doing the homology analysis: the intergenic sequences differ, but the LRAE is similar

The results are insensitive to parameter

perturbation

More examples supporting the existence of LRAE :

Sorghum v.s. Rice : Sh2/A1 (homologous locu

s)

SM0000063 SM0000064 SM0000065 SM0000066 SM0000067 SM0000068 SM0000069 SM0000070 SM0000071

SM0000032 SM00000170Sorghum v.s. Maize : Adh1 (homologous locus)

SM0000011 SM0000012hsIFNA

SM0000023 SM0000028 SM0000029IGF2

IFNA2 SM0000074 SM0000075

SM0000080 SM0000081cspB

S/MARt-DB is still under construction. Meaningful statistics should be given when there are enough records available.

It’s possible to reveal LRAE by sequence analysis combined with mechanics investigation in a bioinformatic way

LRAE is hopefully an effective regulatory mechanism in gene transcription

(e.g. SMAR can function via LRAE)

Summary

Thanks For Your Attention !