Linker histones: global and specific functions

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1 Linker histones: global and specific functions Andrzej Jerzmanowski Zakład Biologii Molekularnej Roślin Uniwersytet Warszawski i Instytut Biochemii i Biofizyki PAN

2 Linker (H1) histones basic characteristics 30 nm fiber They are much more abundant than chromatin remodeling proteins It is not true that they are non-essential in higher multicellular organisms They are evolutionary very old and have wittnessed the separation of lineages leading to plants and animals They occur in multiple variant forms Brown D. T. Nature structural & molecular biology

3 LINKER HISTONES? What is a primary, general role of H1 in chromatin structure/function? What are H1 variants for?

4 A little bit of history Jerzmanowski, A. and Maleszewski, M. (1985) Phosphorylation and methylation of Physarum histone H1 during mitotic cycle. Biochemistry 24, Jerzmanowski, A. and Krężel, A.M. (1986) Intramolecular localization and effect on conformational stability in vitro of irreversible interphase phosphorylation of Physarum histone H1. Biochemistry 25, Jerzmanowski, A. and Moraczewska, J. (1988) Distribution of postsynthetic methylation sites in Physarum histone H1. Molec. Biol. Rep. 13, Jerzmanowski, A. and Cole, R.D. (1990) Flanking sequences of Xenopus 5S RNA genes determine differential inhibition of transcription by H1 histone in vitro. J. Biol. Chem. 265, Jerzmanowski, A. and Cole, R.D. (1992) Partial displacement of histone H1 from chromatin is required before it can be phosphorylated by mitotic H1 kinase in vitro. J. Biol. Chem. 267, Prymakowska-Bosak, M., et al.,(1996) Histone H1 overexpressed to high level in tobacco affects certain developmental programs but has limited effect on basal cellular functions. Proc. Natl. Acad. Sci. USA 93, Tomaszewski, R. and Jerzmanowski A. (1997) The AT-rich flanks of the oocyte-type 5S RNA gene of Xenopus laevis act as a strong local signal for histone H1-mediated chromatin reorganization in vitro. Nucleic Acids Research 25, Tomaszewski, R.et al., (1998) Both the 5S rrna gene and the AT-rich flanks of Xenopus laevis oocyte-type 5S rdna repeat are required for histone H1-dependent repression of pol III-type genes in in vitro reconstituted chromatin. Nucleic Acids Research 26, Prymakowska-Bosak, M., et al.. (1999) Linker histones play a role in male meiosis and the development of pollen grains in tobacco. The Plant Cell 11, Jerzmanowski, A., et al.,(2000) Linker Histones and HMG1 Proteins of Higher Plants. Plant Biology 2, Kaczanowski, S. and Jerzmanowski, A. (2001) Evolutionary Correlation Between Linker Histones and Microtubular Structures. J. Mol. Evol. 53, Jerzmanowski, A. (2004) The linker histones. In: Chromatin Structure&Dynamics: State-of-the-Art (Zlatanova, J. and Leuba, S. eds.) Elsevier Brzeski,J. and Jerzmanowski, A. (2004) Plant chromatin epigenetics linked to ATP-dependent remodeling and architectural proteins. FEBS Lett. 567, Wierzbicki, A.T. and Jerzmanowski, A. (2005) Suppression of histone H1 genes in Arabidopsis thaliana results in heritable developmental defects and stochastic changes in DNA methylation. Genetics 169, Jerzmanowski, A. (2007) SWI/SNF chromatin remodeling and linker histones in plants. Biochim. Biophys. Acta 1769,

5 Placement of H1 in respect to nucleosome dyad and DNA Zhou et al., 1998

6 Linker histones - function Linker histones (LH) are critical for the formation of higherorder chromatin structure. According to the current paradigm, the topology of the 30nm chromatin fiber and the degree of its compactness is regulated by LH abundance.

7 Three-domain structure of histone H1 N-tail Globular domain C-tail GH1 Extremely long Long Enormous potential for posttranslational modifications

8 Problem wariantów H1 A list of known mammalian linker histone subtypes Cell Type H1 variants Distinctive characteristics H1.0 Restricted to terminally differentiated cells H1.1 Restricted to certain tissues H1.2 Highest turnover rate, not restrict. to S-phase Somatic H1.3 Highest levels of expression during S-phase H1.4 Turnover rate varies, depending on cell type H1.5 Highest PO 4 levels at all stages H1.X Identified solely in cultured cells Testis-specfic H1t H1t2 HILS1 Oocyte-specific H1oo

9 Current wisdom about H1 variants function Linker histone subtypes are differential organizers of chromatin, rather than general repressors. Clausell et al., PLOS One (2009) Linker histone variants control chromatin dynamics during early embryogenesis (we propose a model that holds that maternally expressed linker histones are key molecules specifying nuclear dynamics with respect to embryonic totipotency). Saeki et al., PNAS (2005)

10 Arabidopsis is an ideal model system to study the function of linker histone variability

11 Isoforms (non-allelic variants) of linker histones in Arabidosps geneome Wierzbicki & Jerzmanowski Genetics, 2005

12 Linker histones of plants H1-1 DICOTS H1-2 MONOCOTS H1-3 DROUGHT INDUCIBLE HMG I/Y - TYPE GREEN ALGAE Jerzmanowski et al. Plant Biol. 2000

13 (A) Phylogenetic tree of Arabidopsis proteins possessing the GH1 domain. (B) Part of the histone H1 branch of the Arabidopsis GH1 tree including Nicotiana tabacum histone H1 variants (NTH1). Trees were built with PHYLIP (Felsenstein 2005) using the maximum parsimony method. Kniżewski et al. unpub.

14 Sequence features of Arabidopsis and Nicotiana tabacum histone H1 variants stress-inducible stress-inducible stress-inducible Kniżewski et al. unpubl.

15 DNA binding sites found experimentally in murine H1 o (Brown et al., 2006) and predicted for Arabidopsis (H1-1, H1-2, H1-3) and Nicotiana tabacum (H1A-F) histone H1 variants Blue residues structurally equivalent to those in H1 0 Red - additional DNA binding residues H1-3/H1C in site 1 no equivalent of Arg74; in site 2, Lys41/71 instead of Arg42 Altered properties of H1-3/H1C GH1 domain may seriously affect the strength of DNA binding Kniżewski et al. unpubl.

16 Translational fusions of Arabidopsis H1 variants with EGFP reporter ATG EGFP H1-1 locus ATG EGFP H1-2 locus ATG EGFP H1-3 locus Puzio M. et al. unpubl.

17 Woreczek zalążkowy zygota globularny późny globularny 2 liścienie, merystem w. pędu (ad) hypokotyl (cd) korzeń (cd) merystem wierzch., czapeczka (hy) przejściowy późny sercowaty siewka Co najmniej do stadium 8-komórkowego zarodek A. thaliana nie wyraża żadnego wariantu H1

18 Fluorescence Recovery after Photobleaching (FRAP) allows studying the dynamics of proteins in vivo The rate of fluorescence recovery reflects the rate at which fluorescent molecules exchange with the photobleached molecules; Before bleaching After bleaching The exchange rate is directly proportional to the rate of diffusion during migration through the nucleus and inversly proportional to the time the molecule resides at the immobile binding site. Partial recovery Complete recovery

19 GFP exchange is determined only by rate of diffusion H2B exchange is determined by stable binding Puzio et al. unpubl.

20 Multiple populations of histone H1 are evident in FRAP recovery curves Stably bound pool Raghuram et al., 2009

21 relative intensity A Prebleach Bleach 0.2s 5s 33.7s 133.7s 253.7s B Complete recovery: H1.3 ~ 33.7s H1.1 ~133.7s for H1.2 weak recovery >300s. 1.2 Prebleach Bleach 0.2s 5s 33.7s 133.7s 253.7s C Prebleach Bleach 0.2s 5s 33.7s 133.7s 302.6s Recovery time of H1.1, H1.2, H1.3 in nuclei of A. thaliana guard cells H1-2-GFP Stable-binding component? 1,4 1,2 1 0,8 0,6 0,4 0,2 0 H1-2 H1-1 H Recovery time (s) Puzio et al. unpublished

22 Arabidopsis H1-3, stability of down regulation by mirna, effect of ABA H1-3 Actin Col-0/h1-3miR Col-0 Col-0 ABA Col-0/h1-3miR ABA ABA up-regulates H1-3 Rutowicz et al., unpubl.

23 Schematic representation of the promoter regions of genes encoding Arabidopsis histone H1 somatic variants. Kniżewski et al. unpub. For each promoter the two opposing strands (+/-) are shown. Orange rectangles mark the location of ABRE motifs. The promoters are drawn approximately to scale.

24 Specyfika promotora H1-C z tytoniu To further characterize H1C we cloned a 350 bp fragment of its promoter using the Genome-Walker approach. Besides the sequence elements typically present in eukaryotic promoters, the H1C promoter contains a number of potential regulatory motifs (Figure 2a). The GT-box (Zhou, 1999) and the CACGTG (Foster et al., 1994) motifs are found in promoters regulated by light and other stimuli. The latter sequence and the CE-1 motifs are responsible for the induction by ABA (Shen and Ho, 1995; Hobo et al., 1999). The H1C promoter also contains the sucrose response element SURE-2 (Grierson et al., 1994) and the H1-box (residues -247 to -253) characteristic of all metazoan and some plant histone H1 genes (Dalton and Wells, 1988). Przewłoka, M. et al., Planta (2002) 215:

25 Localization of H1 variants in Arabidopsis H1.1 H1.2 H1.3 Puzio J. et al., unpublished

26 Merystemy korzeni bocznych 3 tygodniowych siewek roślin H1.3 EGFP hodowanych w warunkach dnia długiego: 16 godz. dzień/8 godz. noc Merystemy korzeni bocznych 3 tygodniowych siewek roślin H1.3 EGFP hodowanych w warunkach stresu braku światła (3 doby w ciemności) % korzeni zaindukowanych stresem braku światła w stosunku do nie zaindukowanych, w odniesieniu do kontroli: ekspresja GFP brak ekspresji GFP wszystkie H1-3 (kontr) H1-3 (ciem.) H1-2 (kontr) H1-2 (ciem) H1-1 (kontr) H1-1 (ciem) 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% H1.1EGFP H1.2EGFP H1.3EGFP brak ekspresji GFP ekspresja GFP Puzio J. et al., unpublished

27 Indukcja H1-3 w primordiach korzeni bocznych po 72 godz. w ciemności Normalny fotoperiod Po 72 godz. w ciemności Fluorescencja Primordia korzeni bocznych Światło przechodzące

28 Zmiana obsadzenia wybranych loci przez warianty H1 normal (T0) vs. low light (T1) 100% 100% 90% 90% 80% 80% 70% 70% 60% 50% 40% 30% H1.2 T0 H1.3 T0 H1.1 T0 60% 50% 40% 30% H1.2 T1 H1.3 T1 H1.1 T1 20% 20% 10% 10% 0% H1-3 Ta3 Aktyna AtSN Internal UBQ10 0% H1-3 Ta3 Aktyna AtSN Internal UBQ10

29 Przykładowe zdjęcie doniczki z kontrolą: rośliny dzikie (wt) i mutanty (h1.3) są tej samej wielkości.

30 Kontrola: średnice (w cm) rozet 4-tygodniowych roślin o fenotypie dzikim (wt) i mutantów h1.3 hodowanych w warunkach z ciągłym nawilżaniem gleby (zaznaczono odchylenie standardowe).