Download with epiC - Beckman Coulter Česká republika sro

Honba za určením komplexní
epigenetické značky
(aneb průkaz jaderného aktinu?)
Ivan Raška
Ústav buněčné biologie a patologie,1. LF UK v Praze
Buněčná analýza v biomedicíně, 8. června 2016
Tento seminář se koná ve spolupráci s firmami Beckman Coulter Česká
republika s.r.o., SIGMA-ALDRICH spol. s r. o., součást Merck KGaA,
a OLYMPUS CZECH GROUP, S.R.O.
Complexity
of the cell
nucleus
Thin sectioned
hepatocyte
labeled (5 nm
gold) for DNA
as seen in the
electron
microscope
If we compare the size of a human cell nucleus encompassing 46
chromosomes to a living room of 20m2, then - by analogy - it is
necessary to pack in such a “room” 46 thin ropes (2 mm in diameter)
with the total length of 2000 km. Chromatin is highly dynamic and it is
not yet known how DNA is packed in nuclei of living cells.
The cell nucleus is a repository of the genome. The
nucleus is the most complicated cell organelle and
the packing of DNA is but the initial problem. The
specific segments of the genome (such as genes) are
under highly regulated regime transcribed into
RNA, RNA molecules are processed (e.g. splicing of
pre-mRNA) and move to their site of action, or are
degraded. DNA is under surveillance of repair
mechanisms (20-50 thousands potentionally
harmful events per cell/day). And within the
context of my talk, DNA has to be once and
only once replicated during the cell cycle.
Mitotic chromosomes and interphase chromosome territories
3 pairs of chromosomes
(chromosome territories)
are FISH visualized
in 3 different colors
Spread mitotic chromosomes
Metaphase (M)
Cell cycle
Chromosome territories
Interphase (G1, S, G2)
Note a decondensed
structure of
chromosome territories
with respect to mitotic
chromosomes
Scene and performing players on the stage
•
•
•
•
•
•
•
•
•
•
Human diploid cells, actin, histones
Cell cycle and replication, epigenetic modifications
Bio-dUTP (biotinylated base analogue of thymine as replication marker)
Antibody to biotin (ab to biotin)
Monoclonal antibody to actin ( monoclonal ab to actin)
Ab to PCNA (Proliferation Cell Nuclear Antigen used as replication marker)
Secondary abs bearing various fluorochromes
YOYO (fluorescent dye staining nucleus)
Biochemical/immuno(cyto)chemical techniques
Synthesized blocking peptides (used for depletion of abs)
This being said,
let´s start with the story of human
diploid fibroblasts (results valid for
other tested cell types as well) and the
commercial monoclonal antibody
to actin .....
RT
Anti-actin
YOYO
As chance may frequently lead
to unexpected and interesting findings...
COLD-DEPENDENT DETECTION
Anti-actin
+4°C
YOYO
....some nuclei exhibit bright signal at low T.
The corresponding epitope was called epiC.
Straightforward questions raised:
WHICH NUCLEI? Why only some of
the nuclei were positive?
WHY COLD? Does it protect
antigen/epiC from degradation?
WHICH NUCLEAR STRUCTURES?
Chromatin?
(Same results obtained with bio-dUTP instead of PCNA)
anti-actin
PCNA
very early S
early S
merged
anti-actin
PCNA
mid S
late S
merged
anti-actin
PCNA
G2
M
merged
Epitope positivity disappeared
during the early G1
anti-actin
EpiC detection
was cell Why
cycleonly
dependent:
WHICH NUCLEI?
some of
theinnuclei
were positive?
Positive
“S-to-early
G1” cell cycle window
WHY COLD? Does it protect
antigen/epiC from degradation?
WHICH NUCLEAR STRUCTURES?
Chromatin?
Was the Epitope Degraded
at RT ?
anti-actin
2hr RT, +Cy3
merged
anti-actin
O/N 4°C, +AlexaFluor488
Secondary antibody stabilizes the antibody-epitope complex!
Epitope was not degraded at RT,
but hydrophobic interactions significantly
decrease as the temperature decreases
Cold-induced structural changes:
cold-induced reversible changes
in higher order protein structure
ARE THE CHANGES REVERSIBLE ?
Temperature-Induced Changes
epiC+actin
PCNA
1 hr RT → O/N 4°C → 2 hrs RT → 3 hrs 4°C →
are reversible!
(Example of mitotic
cells documented here)
→ 2 hrs RT → 3 hrs 4°C
“S-to-early G1” cell cycle window
WHICH NUCLEI? Why only some of
EpiC
detection
waspositive?
cell cycle dependent
the
nuclei were
New phenomenon
WHY COLD? Does it protect
of temperature-induced
reversible
changes of
antigen / epitope from
degradation?
in situ epiC immunodetectability
WHICH NUCLEAR STRUCTURES?
Chromatin?
Co-localization of epiC with replication markers
Detailed cross-correlation analysis was necessary:
Pearson’s correlation coefficient
~
rP 
 ( R  R )(G  G )
 ( R  R )  (G  G )
i
i
i
2
i
i
i
i
Ri , G i
red (green) fluorescence
intensity in each pixel
R, G
red (green) average
fluorescence intensity
2
r   1;1
rp>0, co-localization, rp~0, no correlation, rp<0, exclusion
Manders EMM, Verbeek FJ and Aten JA (1993), Journal of Microscopy 169:375-382.
PCNA (bio-dUTP)
very early S
early S
actinPCNA(bio-dUTP)
correlation
PCNA (bio-dUTP)
mid S
late S
actinPCNA(bio-dUTP)
correlation
biotin-dUTP (chase)
very early S
early S
actindUTP (chase)
correlation
biotin-dUTP (chase)
mid S
late S
actindUTP (chase)
correlation
“S-to-early G1” cell cycle window
WHICH NUCLEI? Why only some of
EpiC
detection
waspositive?
cell cycle dependent
the
nuclei were
New phenomenon
WHY COLD? Does it protect
of temperature-induced
reversible
changes of
antigen / epitope from
degradation?
in situ epitope immunodetectability
Chromatin structures, the DNA of which was
WHICH
NUCLEAR
STRUCTURES?
replicated
in early
S phase
(=transcriptionally
Chromatin?
competent part of DNA),
co-localized (with a delay!) with epiC
What is the molecular nature of epi C?
(Are we immunodetecting
nuclear actin?)
The establishment of the nature of epiC
represented „a long way to Tipperary. “
Finally, we were able to identify epiC to reside
within histones. Only this then led, by means
of immuno(cyto)chemistry, to the
establishment of the nature of epiC using
various histone preparations and a large
battery of synthesized (blocking) peptides.
EpiC is contained in the histone H4 fraction of acid
extracted nuclear histones separated by reverse
phase HPLC
• C - the zoomed part of
chromatogram containing
fractions of core histones.
• D - SDS-PAGE of the
marked fractions
containing from left to
right H2B, H2B/H2A,
H2A, H4, markers, H2A,
H2A, H3 .
• E - immunoblots/marker
blots corresponding to gels
in D with only histone H4
fraction exhibiting
positivity (arrow).
Lanes 1-5:
gels stained after
blotting
Actin: 1,4,1´,4´
Histones: 2,5,2´, 5´
Lanes 1´- 5´:
Western blots
Acid extracted nuclear histones contain epiC
Acid extracted nuclear histones and rabbit muscle actin
were probed with anti-actin antibody. Lanes A2´ and
B5´ show epiC positivity (arrows).
With regard to RT or 4oC temperatures, two complementary experiments were performed in A and B.
Roche histones:
lanes 2, 2´
Recombinant histone:
lanes 3,4, 3´,4´
(different loadings)
Anti-actin ab recognized epiC in histone H4 of
Roche histones, but not in recombinant histone H4
SDS/PAGE (Figure - left part) and immunoblotting with anti-actin
at 4°C (Figure - right part) is shown. Commercial calf thymus
histone preparation (Roche histones) is commonly used as a control
sample containing posttranslationally modified histones, while
recombinant H4 histones do not contain modifications.
Dual epigenetic
modification of
lysines (position
16 and 20).
Note that the original immunogenic
sequence of actin
differs from the
relevant amino acid
sequence of
histone H4
Anti-actin ab recognized
selectively at 4°C, but not at
RT, the peptide bearing
H4K16ac-K20me2 postranslational modifications.
Blocking tests with peptides
were performed in situ (A)
and in in vitro dot blots (B).
Only the peptide sp473a,
bearing H4K16ac-K20me2,
exhibited the epiC signature.
sp455 bears K16ac
sp466 bears K16ac
sp472 bears K16ac-K20me1
Summary
• EpiC nuclear epitope found surprizingly via anti-actin ab labeling at
low T
• EpiC seen in the cell cycle window S – early G1 phase
• A new phenomenon of temperature-induced reversible changes of in
situ epitope immunodetectability established
• Chromatin structures, the DNA of which was replicated in early S
phase, co-localized (with a delay!) with epiC
• By means of immuno(cyto)chemistry, epiC identified with lysine K16acetylated and lysine K20-dimethylated (H4K16ac-K20me2) within
histone H4 tail
• Identified dual epigenetic marker has to play play an
important role in the maintenance/transfer of epigenetic
information on transcriptionally competent part of
genome
• Even a cross-reacting antibody can make you sometimes happy
The Multi-Partite Epigenetic Marker
Involved in DNA Replication
Thanks for your attention
I would like to thank my collaborators Helena Fidlerová, Jana
Kalinová, Miroslava Blechová and Jiří Velek.
Supported by GAČR grant P302/12/G157 ,
Charles University grants UNCE 204022 and Prvouk/1LF/1
Complexity
of the cell
nucleus
Thin sectioned
hepatocyte
labeled (5 nm
gold) for DNA
as seen in the
electron
microscope
If we compare the size of a human cell nucleus to a living
room of 20m2, then - by analogy - it is necessary to pack in
such a “room” 46 thin ropes (2 mm in diameter) with the total
length of 2000 km. Chromatin is highly dynamic and it is not
yet known how DNA is packed in nuclei of living cells.
Complexity of the cell nucleus
Thin
sectioned
hepatocyte
seen in the
electron
microscope
If we compare the size of a human cell nucleus to a living
room of 20m2, then - by analogy - it is necessary to pack in
such a “room” 46 thin ropes (2 mm in diameter) with the total
length of 2000 km. Chromatin is extremely dynamic and it is
not yet known how DNA is packed in nuclei of living cells.
Summary
• Surprizingly, by means of the commercial anti-actin antibody the
epigenetic marker epiC was identified.
• The epiC marker was detected in the cell
• By means of immunochemistry and immunocytochemistry, epiC
identified with K16-acetylated and K20-dimethylated (H4K16acK20me2) within histone H4 tail. This mark appears, but with a delay,
only on early replicated (=transcriptionally competent) part of the
genome.
• Identified dual, cell cycle dependent epigenetic marker
epiC has to play an important role in the
maintenance/transfer of epigenetic information on
transcriptionally competent part of genome