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Chromatin regulation by post-translational modification
of non-histone proteins
Klaus D. Grasser
Department of Biotechnology, Institute of Life Sciences
Aalborg University, Denmark
How is the genomic DNA actually packaged
into eukaryotic chromatin?
DNA + histones + non-histones
 functional consequences of packaging
 controlling DNA-accessibility
approx. 2 m of genomic DNA has to fit
into a nucleus of approx. 10 m !
High Mobility Group (HMG) Proteins
traditional definition:
 chromosomal non-histone proteins (9-28 kDa)
 extractable from chromatin with ~0.35 M NaCl
 soluble in 2% TCA or 5% PCA
 high content of basic and acidic amino acid residues

in higher plants: HMGA and HMGB proteins
Plant HMGB proteins
 5 different HMGB proteins per species
DNA
 non-sequence-specific DNA-binding
 recognition of DNA structures
 DNA-bending and supercoiling activity
 formation of nucleoprotein structures
++++ +
++
HMG
Box
HMGB (13-20 kDa)
-------
HMG-box domain
Mass data of HMGB1 and HMGB2/3 isolated from
maize BMS suspension culture cells
HMGB1
HMGB2
HMGB3
a The
calc. Massa
untreatedb
APb,c
17145.9
15315.7
15007.3
17531
15556
15326+15406
17148
15318
15169
mass values (in Da) were calculated based on the known protein sequences.
masses (in Da) of native HMGB proteins were determined by nanospray mass spectrometry on the ion trap LC-Q.
cAP, dephosphorylation of native HMGB proteins by treatment with alkaline phosphatase.
dNumber of phosphorylations determined by dephosphorylation of native HMGB proteins by AP.
bThe
phosphorylationsd
4
3
2+3
Zm-HMGB1
Zm-HMGB2
Zm-HMGB3
Zm-HMGB4
Zm-HMGB5
M1
│
MKGAKSKGAAKADAKLAVKSKGAEKPAKGRKGKAGKDPNKPKRAPSAFFVFMEEFRKEFKEKNPKNKSVAAVGKAAGDRWKSL
MKGKADTSKKDEGRLRAG.GAAGKRKKAAASGKPKRPPSAFFVFMSEFRQEYQALHPGNKSVATVSKAAGEKWRAM
MKGKANASKKDEARLRAGGGGAGKRKKAAASGKPKRPPSAFFVFMSEFRQEYQAQHPGNKSVAAVSKAAGEKWRSM
MKSRARSTAGDSRLSVRKTKAEKDPNKPKRPPSAFFVFMEEFRKDYKEKHPNVKQVSVIGKAGGDKWKSL
MKDTSFKATGAKRKKVGGAKRGLTPFFAFLAEFRPQYLEKHPELKGVKEVSKAAGEKWRSM
K123
D134
E157
│
│
│
SESDKAPYVAKANKLKLEYNKAIAAYNKGESTAAKKAPAKEEEEEDEEESDKSKSEVNDEDDEEGSEEDEDDDE
SDQEKQPYVDQAGQKKQDYEKTKANFDKKESTSSKKAKTEDEDGSKSEVDDEDGSSDEENDDDE
SEQEKQPYVDQAGQKKQDYEKTKANIEK..STSSKKAKTDDDDGSKSEVDDEDGGSDEDNDDDE
SDAEKAPYVSKAEKLKAEYTKKIDAYNNKQSGDPTASGDSDKSKSEVNDEDEEGDE
SDEEKAKYGSSKKQDGKASKKENTSSKKAKADVREGDEAEGSNKSKSEVEDDEQDGNEDEDE
CK2 phosphorylation sites of the maize HMGB proteins
(as determined by mass spectrometry of tryptic peptides
derived from native and in vitro phosphorylated HMG proteins)
aa157
aa139
aa138
aa126
aa123
Some effects of the phosphorylation of HMGB proteins by CK2
 reduced affinity for linear dsDNA
 no effect on the recognition of DNA minicircles, but different complexes formed
 affinity for mononucleosomes unchanged
 stabilisation of the proteins against thermal denaturation
 increased activity in stimulating site-specific  recombination
 interaction with the transcription factor Dof2 abolished
+
no protein interaction
AP
+ AP
+
protein interaction
Architectural proteins (AP) facilitate the formation
of complex nucleoprotein structures
AP
Future directions
systematic analysis of post-translational modifications of all HMGB proteins
including other chromatin-associated proteins such as HMGA, SSRP1, CDC68
functional consequences of the modifications (chromatin structure, transcription, etc.)
identification of the enzymes catalysing the modifications (protein kinases etc.)
regulatory signalling networks controlling the modifying enzymes
Genomics/Proteomics
Signaltransduction
Function
(chromatin, transcription etc.)
Plant chromatin-associated proteins
http://www.bio.auc.dk/
Aalborg University
Meg Crookshanks
Jeanette R. Gade
Jesper T. Grønlund
Nicholas M. Krohn*
Dorte Launholt
Diana J. Leeming
Jacek Lichota*
Hanne Krone Nielsen
Christian Stemmer*
Malene Thompsen
Klaus D. Grasser
CNB, CSIC, Madrid
Silvia Fernández
Gema Lopez
Juan C. Alonso
Tokyo University
Shuichi Yanagisawa
Hexal BioTech, München
Rudi Grimm
Peter Fojan
Guy Bauw