Chromatin regulation by post-translational modification

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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
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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 !
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• 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

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• Plant HMGB proteins
•  
• ?5 different HMGB proteins per species
• non-sequence-specific DNA-binding 
• recognition of DNA structures
• DNA-bending and supercoiling activity
• formation of nucleoprotein structures
•  

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Mass data of HMGB1 and HMGB2/3 isolated from
maize BMS suspension culture cells   calc.
Massa untreatedb APb,c phosphorylationsd HMGB1 1
7145.9 17531 17148 4 HMGB2 15315.7 1
5556 15318 3 HMGB3 15007.3 153261540
6 15169 23 a The mass values (in Da)
were calculated based on the known protein
sequences. bThe 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.
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      M1



Zm-HMGB1 MKGAKSKGAAKADAKLAVKSKGAEKPAKGRKGKAGKDPN
KPKRAPSAFFVFMEEFRKEFKEKNPKNKSVAAVGKAAGDRWKSL
Zm-HMGB2 MKGKADTSKKDEGRLRAG.GAAGKRKKAAASG
KPKRPPSAFFVFMSEFRQEYQALHPGNKSVATVSKAAGEKWRAM
Zm-HMGB3 MKGKANASKKDEARLRAGGGGAGKRKKAAASG
KPKRPPSAFFVFMSEFRQEYQAQHPGNKSVAAVSKAAGEKWRSM
Zm-HMGB4 MKSRARSTAGDSRLSVRKTKAEKDPN
KPKRPPSAFFVFMEEFRKDYKEKHPNVKQVSVIGKAGGDKWKSL
Zm-HMGB5
MKDTSFKATGAKRKKVGGAKRGLTPFFAFLAEFRPQYLEKHPELKGVKEV
SKAAGEKWRSM    
K123 D134
E157

SESDKAPYVAKANKLKLEYNKAIAAY
NKGESTAAKKAPAKEEEEEDEEESDKSKSEVNDEDDEEGSEEDEDDDE
aa157 SDQEKQPYVDQAGQKKQDYEKTKANFDKK
ESTSSKKAKTEDEDGSKSEVDDEDGSSDEENDDDE
aa139 SEQEKQPYVDQAGQKKQDYEKTKANIEK..ST
SSKKAKTDDDDGSKSEVDDEDGGSDEDNDDDE
aa138 SDAEKAPYVSKAEKLKAEYTKKIDAYNNKQSGDP
TASGDSDKSKSEVNDEDEEGDE
aa126 SDEEKAKYGSSKKQDGKASKKENTSSKKAKADVR
EGDEAEGSNKSKSEVEDDEQDGNEDEDE
aa123
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)
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• 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
•  

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Architectural proteins (AP) facilitate the
formation of complex nucleoprotein structures
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• 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.)
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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
Peter Fojan Malene Thompsen Guy
Bauw Klaus D. Grasser CNB, CSIC, Madrid
Silvia Fernández Gema Lopez Juan C.
Alonso Tokyo University Shuichi
Yanagisawa Hexal BioTech, München Rudi
Grimm