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Chromatin regulation by post-translational modification of non-histone proteins Klaus D. Grasser

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

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Chromatin regulation by post-translational modification of non-histone proteins Klaus D. Grasser

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  1. Chromatin regulation by post-translational modification of non-histone proteins Klaus D. Grasser Department of Biotechnology, Institute of Life Sciences Aalborg University, Denmark

  2. 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 !

  3. 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

  4. DNA HMG-box domain • 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

  5. Mass data of HMGB1 and HMGB2/3 isolated from maize BMS suspension culture cells calc. Massa untreatedbAPb,c phosphorylationsd HMGB117145.91753117148 4 HMGB215315.71555615318 3 HMGB315007.315326+1540615169 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.

  6. 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 peptides derived from native and in vitro phosphorylated HMG proteins)

  7. 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

  8. AP AP + AP + + no protein interaction protein interaction Architectural proteins (AP) facilitate the formation of complex nucleoprotein structures

  9. 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.)

  10. 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, MadridSilvia Fernández Gema Lopez Juan C. Alonso Tokyo UniversityShuichi Yanagisawa Hexal BioTech, MünchenRudi Grimm

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