Chromatin regulation by post-translational modificationChromatin regulation by post-translational modificationof non-histone proteins of non-histone proteins

Klaus D. GrasserKlaus D. GrasserDepartment of Biotechnology, Institute of Life SciencesDepartment of Biotechnology, Institute of Life Sciences

Aalborg University, DenmarkAalborg University, Denmark

How is the genomic DNA actually packaged

into eukaryotic chromatin?

approx. 2 m of genomic DNA has to fit into a nucleus of approx. 10 m !

DNA + histones + non-histones

functional consequences of packaging

controlling DNA-accessibility

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

non-sequence-specific DNA-binding 

recognition of DNA structures

DNA-bending and supercoiling activity

formation of nucleoprotein structures 

HMGBox

+ + + + + + + - - - - - - -

HMGB (13-20 kDa)

DNA

HMG-box domain

Mass data of HMGB1 and HMGB2/3 isolated from maize BMS suspension culture cells  

calc. Massa untreatedb APb,c phosphorylationsd

HMGB1 17145.9 17531 17148 4HMGB2 15315.7 15556 15318 3HMGB3 15007.3 15326+15406 15169 2+3

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.

 

   M1 │ Zm-HMGB1 MKGAKSKGAAKADAKLAVKSKGAEKPAKGRKGKAGKDPNKPKRAPSAFFVFMEEFRKEFKEKNPKNKSVAAVGKAAGDRWKSL Zm-HMGB2 MKGKADTSKKDEGRLRAG.GAAGKRKKAAASGKPKRPPSAFFVFMSEFRQEYQALHPGNKSVATVSKAAGEKWRAM Zm-HMGB3 MKGKANASKKDEARLRAGGGGAGKRKKAAASGKPKRPPSAFFVFMSEFRQEYQAQHPGNKSVAAVSKAAGEKWRSM Zm-HMGB4 MKSRARSTAGDSRLSVRKTKAEKDPNKPKRPPSAFFVFMEEFRKDYKEKHPNVKQVSVIGKAGGDKWKSL Zm-HMGB5 MKDTSFKATGAKRKKVGGAKRGLTPFFAFLAEFRPQYLEKHPELKGVKEVSKAAGEKWRSM   K123 D134 E157 │ │ │ SESDKAPYVAKANKLKLEYNKAIAAYNKGESTAAKKAPAKEEEEEDEEESDKSKSEVNDEDDEEGSEEDEDDDE aa157 SDQEKQPYVDQAGQKKQDYEKTKANFDKKESTSSKKAKTEDEDGSKSEVDDEDGSSDEENDDDE aa139 SEQEKQPYVDQAGQKKQDYEKTKANIEK..STSSKKAKTDDDDGSKSEVDDEDGGSDEDNDDDE aa138 SDAEKAPYVSKAEKLKAEYTKKIDAYNNKQSGDPTASGDSDKSKSEVNDEDEEGDE aa126 SDEEKAKYGSSKKQDGKASKKENTSSKKAKADVREGDEAEGSNKSKSEVEDDEQDGNEDEDE aa123

CK2 phosphorylation sites of the maize HMGB proteins

(as determined by mass spectrometry of tryptic peptidesderived from native and in vitro phosphorylated HMG proteins)

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 

Architectural proteins (AP) facilitate the formation of complex nucleoprotein structures

+ AP

no protein interaction

+ AP+ AP

protein interaction

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 Plant chromatin-associated proteinsproteins

http://www.bio.auc.dk/http://www.bio.auc.dk/

Aalborg University Meg Crookshanks

Jeanette R. Gade Jesper T. GrønlundNicholas M. Krohn*Dorte LaunholtDiana J. LeemingJacek Lichota* Hanne Krone NielsenChristian Stemmer* Peter

FojanMalene Thompsen

Guy BauwKlaus D. Grasser

CNB, CSIC, Madrid Silvia Fernández

Gema LopezJuan C. Alonso

Tokyo University Shuichi Yanagisawa

Hexal BioTech, München Rudi Grimm