Linker histones: global and specific functions
Andrzej Jerzmanowski
Zakład Biologii Molekularnej Roślin
Uniwersytet Warszawski
i
Linker (H1) histones – basic characteristics
‘30 nm fiber’
Brown D. T. Nature structural & molecular biology
•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
LINKER HISTONES
What is a primary, general role of H1 in
chromatin structure/function?
What are H1 variants for?
A little bit of history
• Jerzmanowski, A. and Maleszewski, M. (1985) Phosphorylation and methylation of Physarum histone H1 during mitotic
cycle. Biochemistry 24, 2360-2367.
• 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, 6495- 6501.
• Jerzmanowski, A. and Moraczewska, J. (1988) Distribution of postsynthetic methylation sites in Physarum histone H1. Molec. Biol. Rep. 13, 97-101.
• 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, 10726-10732.
• 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, 8514-8520.
• 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, 10250-10255.
• 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 , 458-465. • 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, 5596-5601.
• 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, 2317-2329.
• Jerzmanowski, A., et al.,(2000) Linker Histones and HMG1 Proteins of Higher Plants. Plant Biology 2, 586-597.
• Kaczanowski, S. and Jerzmanowski, A. (2001) Evolutionary Correlation Between Linker Histones and Microtubular Structures. J. Mol. Evol. 53, 19-30.
• Jerzmanowski, A. (2004) The linker histones. In: Chromatin Structure&Dynamics: State-of-the-Art (Zlatanova, J. and
Leuba, S. eds.) Elsevier 2004.
• Brzeski,J. and Jerzmanowski, A. (2004) Plant chromatin – epigenetics linked to ATP-dependent remodeling and architectural proteins. FEBS Lett. 567, 15-19.
• 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, 997-1008.
• Jerzmanowski, A. (2007) SWI/SNF chromatin remodeling and linker histones in plants. Biochim. Biophys. Acta 1769,
Placement of H1 in respect to nucleosome dyad and DNA
Linker histones - function
• Linker histones (LH) are critical for the formation of
higher-order 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.
Three-domain structure of histone H1
GH1
Globular domain
N-tail
C-tail
Long Extremely longEnormous potential
for posttranslational
modifications
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
4levels at all stages
•
H1.X Identified solely in cultured cells
•
Testis-specfic H1t
•
H1t2
•
HILS1
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).
• Arabidopsis is an ideal model system to study the function of
linker histone variability
Isoforms (non-allelic variants) of linker histones in Arabidosps geneome
Wierzbicki & Jerzmanowski Genetics, 2005
Jerzmanowski et al. Plant Biol. 2000 DICOTS MONOCOTS DROUGHT INDUCIBLE HMG I/Y - TYPE GREEN ALGAE
Linker histones of plants
H1-1
H1-2
(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).
Kniżewski et al. unpub.
Trees were built with PHYLIP (Felsenstein 2005) using
the maximum parsimony method.
Sequence features of Arabidopsis and Nicotiana tabacum histone H1
variants
Kniżewski et al. unpubl.
stress-inducible
stress-inducible
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
Kniżewski et al. unpubl.
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
Blue – residues structurally equivalent to those in H1
0ATG
H1-1 locus
EGFP
ATGH1-1 locus
EGFP
EGFP
ATGH1-2 locus
EGFP
ATGH1-2 locus
EGFP
EGFP
ATGH1-3 locus
EGFP
ATGH1-3 locus
EGFP
EGFP
Translational fusions of Arabidopsis H1 variants with EGFP reporter
Co najmniej do stadium 8-komórkowego zarodek A. thaliana nie wyraża
żadnego wariantu H1
globularny późny globularny Woreczek zalążkowy zygota
przejściowy późny sercowaty siewka
hypokotyl (cd)
korzeń (cd)
merystem wierzch., czapeczka (hy) 2 liścienie, merystem w. pędu (ad)
Fluorescence Recovery after Photobleaching (FRAP) allows studying
the dynamics of proteins in vivo
Before bleaching After bleaching
Partial recovery Complete recovery
•The rate of fluorescence recovery
reflects the rate at which fluorescent
molecules exchange with the photo-
bleached molecules;
• The exchange rate is directly proportional
to the rate of diffusion during migration
through the nucleus and inversly propor-
tional to the time the molecule resides at
the immobile binding site.
Puzio et al. unpubl.
GFP exchange is determined only
by rate of diffusion
H2B exchange is determined by
stable binding
Multiple populations of histone H1 are evident in FRAP recovery curves
Raghuram et al., 2009
Stably bound
pool
0 0,2 0,4 0,6 0,8 1 1,2 1,4 0 100 200 300
Recovery time (s)
rel
ativ
e i
ntensi
ty
H1-2 H1-1 H1-3 A Prebleach Bleach 0.2s 5s 33.7s 133.7s 253.7s Prebleach Bleach 0.2s 5s 33.7s 133.7s 253.7s B C Prebleach Bleach 0.2s 5s 33.7s 133.7s 302.6sRecovery time of H1.1, H1.2, H1.3 in nuclei of A. thaliana guard cells
Complete
recovery:
H1.3 ~ 33.7s
H1.1 ~133.7s
for H1.2 weak
recovery >300s.
H1.3
H1.1
H1.2
Puzio et al. unpublished
Stable-binding component?
Arabidopsis H1-3, stability of down regulation by miRNA, effect of ABA
H1-3
Col-0/h1-3miR
Col-0
Col-0
ABA
Col-0/h1-3miR
ABA
Actin
ABA up-regulates H1-3
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.
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)”.
H1.1
H1.2
H1.3
Puzio J. et al., unpublished
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) 55 135 190 H1-3 (ciem.) 165 50 215 H1-2 (kontr) 156 13 169 H1-2 (ciem) H1-1 (kontr) 213 - 213 H1-1 (ciem) 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 1 2 3 4 5 6 7 8 brak ekspresji GFP ekspresja GFP
H1.1EGFP
H1.3EGFP
Merystemy korzeni bocznych 3 tygodniowych siewek roślin H1.3 EGFP hodowanych w warunkach dnia
długiego: 16 godz. dzień/8 godz. noc
H1.2EGFP
Indukcja H1-3 w primordiach korzeni bocznych po 72 godz. w ciemności
Normalny fotoperiod
Po 72 godz. w ciemności
Fluorescencja
Światło przechodzące Primordia korzeni bocznych
Zmiana obsadzenia wybranych loci przez warianty H1
normal (T0) vs. low light (T1)
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
H1-3 Ta3 Aktyna AtSN1
152-153 Internal UBQ10 H1.2 T0 H1.3 T0 H1.1 T0 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
H1-3 Ta3 Aktyna AtSN1
152-153
Internal UBQ10
H1.2 T1 H1.3 T1 H1.1 T1
