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Biochimica et Biophysica Acta 1677 (2004) 181–186

Review

Chromatin and cell death

Marco E. Bianchi*, Angelo Manfredi

San Raffaele Scientific Institute, via Olgettina 58, 4 Piano A1, Milan I-20132, Italy

Received 23 September 2003; received in revised form 8 October 2003; accepted 9 October 2003

Abstract

HMGB1, a very mobile chromatin protein, leaks out from necrotic cells and signals to neighbouring cells that tissue damage has occurred.

At least one receptor for extracellular HMGB1 exists, and signals to different cells to divide, migrate, activate inflammation or start an

immune response. Remarkably, apoptotic chromatin binds HMGB1 irreversibly, thereby ensuring that it will not diffuse away to activate

responses from neighbouring cells. Thus, dying cells use their own chromatin to signal how they have died. We argue that the nuclear events

in apoptosis serve to control the molecular signals that dying cells send out.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Chromatin; Histone; HMGB; Apoptosis; Necrosis

1. Introduction

Cells must be able to respond to a variety of cues from

their environment. Among these, information about the

well-being of other cells in the same tissue, and in some

cases in distant areas of the body, is of critical importance.

Cells undergo unprogrammed death or necrosis mainly as a

consequence of mechanical trauma, severe hypoxia, some

types of infection, or poisoning. Cells can also undergo

programmed death or apoptosis: in these cases, cells put an

end to their existence because they have either suffered

irreparable damage, are infected, do not receive appropriate

signals from their environment, or are specifically told to die

by nearby cells. Apoptosis is generally the result of a

decision of the cell involved, and cells that have died this

way often only need to send out an ‘‘eat me’’ signal to

macrophages and other professional or amateur phagocytic

cells. Necrosis, on the other hand, is the consequence of an

unpredictable or uncontrolled event; it has to be communi-

cated to the surrounding cells and the signal must be

amplified to elicit cellular responses to control and repair

the damage.

Surprisingly, it turns out that the signal broadcast by

necrotic cells is their own chromatin. We will review here

the evidence that led to the identification of chromatin

0167-4781/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.bbaexp.2003.10.017

* Corresponding author. Tel.: +39-0226434774; fax: +39-0226434861.

E-mail address: bianchi.marco@hsr.it (M.E. Bianchi).

protein HMGB1 (High Mobility Group B1) as the necrosis

signal, and some of the ways cells use this signal. Finally,

we will argue that the nuclear events of apoptosis (histone

modification and chromatin condensation) have evolved to

control the signalling from the dying cell to its neighbours.

2. HMGB1’s role in the nucleus

HMGB1 (formerly named HMG1 but also known as

amphoterin and sulfoglucuronyl carbohydrate binding pro-

tein, SBP-1) was identified almost 30 years ago; the name

simply indicates that it is a small protein (215 residues) that

runs fast in SDS-polyacrylamide gels [1]. Structurally, it has

two consecutive L-shaped domains (called HMG boxes) and

a 30-amino-acid-long acidic ‘‘tail’’, connected by short

peptides [2].

HMGB1 binds to DNAwithout sequence specificity. Yet

the protein facilitates numerous nuclear transactions, includ-

ing transcription, replication, V(D)J recombination, and

DNA transposition, and interacts with p53, steroid hormone

receptors, NF-nB, homeobox-containing proteins and TBP

[3,4]. HMG boxes bind to the minor groove of B-type DNA,

and upon binding they distort the double helix sharply,

inducing bends of 90j and more. The architectural changes

induced in DNA are believed to facilitate the assembly of

multiprotein complexes at the distorted site.

HMGB1 also binds with relatively high affinity to

already distorted DNA, such as four-way junctions,

M.E. Bianchi, A. Manfredi / Biochimica et Biophysica Acta 1677 (2004) 181–186182

‘‘kinked,’’ undertwisted, and covalently modified DNA [1].

Significantly, it also binds to DNA segments at the entry/

exit of nucleosomes, much in the same way as histone H1.

But whereas H1 is believed to have a general repressing

function, locking in place nucleosomes and rendering them

less accessible and less mobile, HMGB1 can facilitate

nucleosome sliding, at least in vitro [5]. For this to happen,

HMGB1’s interaction with nucleosomes must be highly

reversible: in fact, an HMGB1 truncation lacking the acidic

tail binds to nucleosomes much more tightly and impedes

their sliding.

We have shown that, in living cells, HMGB1 is indeed

the most mobile nuclear protein [6]. GFP was fused to the

acidic C-terminus of HMGB1, and the fusion protein was

shown to have the same activities of unmodified HMGB1

[7]. Photobleaching experiments established that the entire

pool of HMGB1 roams the nucleus: there is no evidence of

a more residential fraction of the protein (Fig. 1). Each

Fig. 1. HMGB1 dynamics in living cells. Photobleaching techniques are

noninvasive microscopy methods that reveal the dynamics underlying the

steady-state distribution of a fluorescently tagged protein in living cells. In

fluorescence loss in photobleaching (FLIP), a small area is repeatedly

bleached with a high intensity laser pulse. After bleaching, the labelled

protein is photochemically altered, so that it no longer fluoresces, but

otherwise retains completely its biological activity. The loss of fluorescence

from outside the bleached region is due to fluorescent molecules that

repopulate the area that was initially bleached. If the fluorescent molecules

are fixed, very few molecules will move under the laser flux and the outside

area will remain fluorescent even after many bleach pulses (a). If a protein

is very mobile, the outside area will be bleached fast (b). The intensity of

total fluorescence in the whole nucleus after each laser pulse can be plotted

(relative to the initial intensity) against time (c). HMGB1-GFP fluorescence

is lost much faster than that of other nuclear proteins, because HMGB1-

GFP is more mobile. HMGB1 has the same mobility in interphase nuclei,

where chromatin is accessible for transcription, and in mitotic chromo-

somes, where chromatin is condensed. Figure adapted from Ref. [6]

individual nucleosome is visited by HMGB1 every 2 s on

average, and the protein will stay there for a small fraction

of a second. This hectic movement ensures that HMGB1

will simply ‘‘wander’’ where it is required within a reason-

able time, do its job, and then leave. In a sense, HMGB1 can

be regarded as the general lubricant of chromatin, or a

‘‘chromatin chaperone’’ that uses no energy besides Brow-

nian motion.

3. A conceptual leap: a chromatin component signals

inflammation

Given the unassailable consensus that HMGB1 is a

nuclear protein, a lot of people were shocked when it

was reported that monocytes and macrophages actually

secrete HMGB1 upon activation, and that secreted

HMGB1 is a mediator of inflammation—in fact, extra-

cellular HMGB1 actually is an inflammatory cytokine.

Excessive extracellular HMGB1 leads to systemic inflam-

mation, sepsis and ultimately even death, both in mice

and in humans [8].

Why should a chromatin protein be secreted? We went

back to our observation that HMGB1 could be passively

released in the medium by detergent-solubilized cells, and

asked ourselves how cells could make themselves per-

meable in a more natural, soap-free situation. The obvi-

ous answer is: necrosis. Necrotic cells lose the integrity

of their membranes, and do leak out all proteins that are

soluble (Fig. 2). It was then easy to prove that wild-type

necrotic cells evoke a strong inflammatory response from

macrophages, whereas Hmgb1� /� necrotic cells cannot

leak out HMGB1 and evoke only a very mild inflam-

matory response [6]. This finding was confirmed in an

animal model: mice were treated with a high dose of the

analgesic acetaminophen (paracetamol) to induce massive

liver cell necrosis, and given anti-HMGB1 antibodies.

The blockage of HMGB1 released by necrotic liver cells

did not protect against liver damage itself (as indicated

by total serum transaminase activity) but did significantly

reduce inflammatory cell recruitment to the damaged liver

(as measured by myeloperoxidase activity, a marker of

neutrophils) [6].

4. An alternative route to extracellular HMGB1: active

secretion

In fact, while for many of us HMGB1 was a nuclear

protein, for others HMGB1—under the name of ampho-

terin—had been for several years a protein located on the

external side of the cell membrane of neuronal cells [9].

Moreover, amphoterin could induce neurite extension, via

the interaction with a membrane receptor, receptor for

advanced glycation endproducts (RAGE) [10]. The iden-

tity between HMGB1 and amphoterin became clear after

Fig. 2. Intracellular and extracellular states of HMGB1. In most types of normal, healthy cells, HMGB1 (small green spheres) is nuclear but undergoes rapid

cycles of binding and detachment from chromatin. When a cell undergoes necrosis, its membranes lose their integrity, HMGB1 is no longer constrained into the

nucleus and passively diffuses out of the cell. On the contrary, when the cell activates its apoptosis program, as a late event its chromatin collapses and HMGB1

becomes tightly attached to the nuclear remnants. Thus, apoptotic cells do not signal their own death, because they retain HMGB1. Resting, non-activated

inflammatory cells, such as monocytes or macrophages, contain HMGB1 in the nuclear compartment. When activated by lipopolysaccharide or inflammatory

cytokines, they translocate the nuclear HMGB1 into the cytoplasm, and from here into specialized organelles, the secretory lysosomes; HMGB1 is then

exocytosed. Thus, HMGB1 can be released either passively by necrotic cells, or actively by inflammatory cells. Extracellular HMGB1 then reaches responsive

cells (green in the drawing), either by diffusion in the immediate vicinity or via the blood stream to more distant compartments. Extracellular HMGB1 binds to

specific receptors (so far, the only validated receptor for HMGB1 is RAGE, but others are likely to exist). Signal transduction through RAGE activates several

responses, depending on the cell type: inflammatory cells are activated, stem cells proliferate, and several cell types migrate towards the source of HMGB1.

Antibodies against HMGB1 abrogate these responses, and also reduce the dispersal of highly metastatic lung carcinoma cells. Figure adapted from Ref. [32]

M.E. Bianchi, A. Manfredi / Biochimica et Biophysica Acta 1677 (2004) 181–186 183

the proteins were sequenced, and the conflict between

being a nuclear and a secreted protein evaporated after it

was found that monocytes actually secrete the HMGB1

molecules stored in the nucleus (and sometimes deplete

the nuclear pool completely) (Fig. 2). The group of Anna

Rubartelli showed that HMGB1 belongs to the small

group of proteins that are not secreted via the endoplas-

mic reticulum, but rather are accumulated directly from

the cytoplasm into specialized organelles of haemato-

poietic cells, the secretory lysosomes [11]. No one knows

how neurons can secrete HMB1, since these contain no

secretory lysosomes.

We recently clarified how monocytic cells relocate

HMGB1 from the nucleus to the cytoplasm [12]: activation

by inflammatory signals shifts the balance towards chroma-

tin acetylation, and HMGB1 becomes hyperacetylated.

Acetylation of two specific clusters of lysines interferes

with nuclear import signals, but not with nuclear export, and

HMGB1 is accumulated in the cytoplasm. Uptake in secre-

tory lysosomes then happens by default.

5. Extracellular HMGB1: a signal for tissue damage

So far, we have discussed how HMGB1 can diffuse

away from cells that have died in an unprogrammed

way, and have pointed out that RAGE is a receptor for

extracellular HMGB1. RAGE is a ubiquitous receptor,

although it is expressed at different levels in different

cells. Thus, HMGB1 can be a signal for tissue damage,

and all cells can decide whether something has to be

done about it. Inflammation is the front-line response to

any type of tissue damage, and in fact monocytic cells

are recruited and start producing inflammatory cytokines

[13,14]. However, professional inflammatory cells also

secrete HMGB1 in an active way, and can create a

loop of activation by HMGB1 and secretion of

HMGB1. We speculate that inflammatory cells have

added HMGB1 secretion to their inflammatory palette

as a later adaptation to reuse a primitive signal of

tissue damage.

Unpublished data (Rovere, Bianchi and Manfredi)

show that another type of myeloid cells, dendritic cells,

responds to extracellular HMGB1 as a signal of danger,

and starts the immune response to antigens that might be

presented by invading microorganisms, or to self-antigens

when microorganisms are absent. This makes sense from

an evolutionary point of view: mammals should expect

infection to follow trauma (like wounds or burns), and it

may be advantageous to activate the immune system

whether microbial antigens are already present or not

yet. However, if infection does not follow trauma, the

M.E. Bianchi, A. Manfredi / Biochimica et Biophysica Acta 1677 (2004) 181–186184

response of the immune system to extracellular HMGB1

may lead to reaction against self antigens and, in patients

with a predisposing genetic background, autoimmune

diseases. In fact, HMGB1 is certainly involved in one

autoimmune pathology, rheumatoid arthritis [15,16], and

antibodies against HMGB1 can ameliorate or cure rheu-

matoid arthritis [17].

Other cell types will also react to HMGB1: vascular

smooth muscle cells migrate [18], endothelial cells up-

regulate the expression of vascular adhesion molecules

[19], intestinal epithelia are disorganized and lose their

barrier function [20]. Remarkably, we have also shown

recently that HMGB1 attracts vessel-associated stem cells

(mesoangioblasts) into damaged tissue, allows their pas-

sage through endothelia, and promotes their proliferation

[31].

HMGB1 is also involved in metastasis [21,22]. Blocking

the HMGB1–RAGE interaction severely reduces the dis-

persal of highly metastatic Lewis lung carcinoma cells in

mice, and even shrinks the primary tumour mass. Perhaps,

tumour cells behave like stem cells, and are mobilized by

extracellular HMGB1; an alternative but not mutually

exclusive explanation is that HMGB1 released by necrotic

cells in the primary tumour increases the permeability of

vessels to tumour cells.

6. Apoptotic cells prevent the release of HMGB1 by

locking it irreversibly to chromatin

The extracellular activities of HMGB1 certainly qualify it

as a potent cytokine, and therefore its release must be tightly

controlled. From this point of view, it is obvious that

apoptotic cells should not release any HMGB1, lest they

activate the same responses of necrotic cells. In fact,

apoptotic cells maintain the integrity of their plasma mem-

branes for several hours, and during this time they are

generally engulfed and disposed either by macrophages or

neighbouring cells. However, if they are not destroyed in

this way, apoptotic cells do lose their membrane integrity

and leak out soluble cellular contents, in a process called

secondary necrosis. We have shown that secondarily ne-

crotic cells still do not broadcast the tissue damage signal,

HMGB1, and do not trigger inflammation [6]. Several

proteins are cleaved by caspases in apoptotic cells, but

apparently HMGB1 is not cut, nor it undergoes other

posttranslational modifications. Rather, HMGB1 binds

tightly and irreversibly to the condensed chromatin of

apoptotic cells. Fluorescence loss in photobleaching (FLIP)

analysis shows that the mobility of HMGB1 in apoptotic

cells is essentially zero: HMGB1 gets unable to diffuse

away from the remnants of the dead cell (Fig. 2). Rather,

apoptotic nuclei represent a sink for extracellular HMGB1:

incubation of permeabilised apoptotic cells with soluble

recombinant HMGB1 results in efficient binding of

HMGB1 to chromatin. Since HMGB1 is not chemically

altered during apoptosis, it must be chromatin itself that gets

modified during apoptosis.

7. Chromatin modifications during apoptosis

One of the hallmarks of apoptosis is that chromatin gets

cleaved to nucleosome-sized fragments. Indeed, this cleav-

age generates a multitude of new DNA ends that are

substrates for DNA chain elongation by terminal deoxynu-

cleotide transferase (TdT) in the popular TUNEL assay.

DNA degradation is a late event in apoptosis, and is effected

by the CAD nuclease, which is activated by caspase-mediate

cleavage of its inhibitory subunit ICAD [23]. However,

inhibition of CAD-mediated DNA degradation does not

abolish the locking of HMGB1 to chromatin [6].

Another long-known modification of chromatin during

apoptosis is its condensation. Chromatin in apoptotic nuclei

is much more condensed than in mitotic chromosomes,

suggesting that nuclear proteins might just get trapped in

collapsed chromatin. However, the mobility of all tested

nuclear proteins (including HMGNs, histone H1, and sev-

eral transcription factors) is not significantly different in the

chromatin of living and apoptotic cells: HMGB1 alone gets

locked in.

Surprisingly, not much more was known until a few

months ago about chromatin modification during apoptosis.

A single report indicated that apoptotic chromatin is under-

acetylated [24], and we found that underacetylation of

histone H4 indeed correlates with HMGB1 locking [6].

Interestingly, exposure of apoptosing HeLa cells to trichos-

tatin A (TSA, a general histone deacetylase inhibitor)

abolished HMGB1 locking and allowed HMGB1 to diffuse

away, much like in necrotic cells. Although this demon-

strates that apoptotic cells can and do signal their demise

like necrotic cells if they are made to shed HMGB1, the

experiment does not prove that histone underacetylation

alone is the cause of HMGB1 locking onto chromatin.

Mammalian cells contain a large amount of underacetylated

heterochromatin, to which HMGB1 should be binding with

high affinity and little mobility if histone acetylation alone is

involved. In fact, HMGB1 does bind heterochromatin [7],

but photobleaching experiments indicate that there is a

single pool of HMGB1 with a single mobility, and therefore

euchromatin and heterochromatin bind HMGB1 in similar

ways. Moreover, HMGB1 does not remain associated to

heterochromatin in detergent-permeabilized cells.

Very recently, a core histone modification has been

identified which is indeed apoptosis-specific: the phosphor-

ylation of the tail of histone H2B at serine 14 [25]. Kinase

Mst1 (mammalian sterile twenty) is activated in apoptotic

cells by cleavage mediated by caspase 3, and is responsible

for phosphorylating H2B S14. Transfection of cells with

truncated Mst1, mimicking the caspase-cleaved form, indu-

ces apoptosis and chromatin condensation, in addition to

H2B phosphorylation. Interestingly, H2B phosphorylation

M.E. Bianchi, A. Manfredi / Biochimica et Biophysica Acta 1677 (2004) 181–186 185

and the H2B N-terminal tail had been known to be essential

for chromatin condensation in Xenopus cell-free systems

[26]. The timing of events during apoptosis caused by

topoisomerase cross-linking to DNA indicates that S14 is

phosphorylated later than the appearance of double stranded-

breaks in DNA, is approximately coincidental with chroma-

tin condensation, and precedes the terminal DNA breakdown

to nucleosome-sized fragments by CAD nuclease.

HMGB1 locking is independent of DNA laddering, and

occurs at the same time as chromatin condensation. Perhaps,

then, H2B S14 phosphorylation is the direct cause of the

irreversible HMGB1 binding to nucleosomes. Where does

this leave the inhibition of HMGB1 locking by trichostatin

A? This result may not be incompatible, if acetylation of

lysine 15 interferes with S14 phosphorylation, or with

HMGB1 binding to phosphorylated S14. The roadmap for

future experimental work appears to be laid out quite

clearly.

8. Apoptosis, or the need (not) to know

The data on H2B phosphorylation provide the first

evidence for a potential apoptotic ‘‘histone code’’, or rather

an apoptotic ‘‘chromatin code’’ if HMGB1 locking is also

considered. However, one wonders why a chromatin code

for death is required at all, and why indeed did apoptosis

evolve.

In multicellular organisms, it is indeed advantageous that

malfunctioning cells, or cells no longer needed, simply die

and are eliminated. Less understandable is why cells should

orchestrate such a complicated series of events as apoptosis.

After all, cells could simply stop functioning, like in

necrosis, and be cleared by professional or amateur phago-

cytic cells. Arguably, a necrosis-style release of lytic

enzymes in the surrounding medium could harm nearby

cells; however, such pollution could be easily avoided by

sealing the plasma membrane and exposing ‘‘eat me’’

signals to accelerate physical removal, as indeed apoptotic

cells do [27]. But why the need for chromatin condensation

and breakdown? We suggest that these processes might have

evolved precisely to let cells distinguish between a benign

and a traumatic demise of their neighbours. If your neigh-

bour dies of old age or cancer, there is not much that you

should or could do; however, if he dies because a car has run

over him, you should probably call the police. Out of

metaphor, the reason for complicated apoptotic manoeuvres

in the nucleus would be simply to avoid signalling trauma if

there is none. We propose that chromatin spillage (in

mammalian cells, HMGB1 spillage) is a simple means to

signal necrosis; absence of chromatin spillage would pre-

vent the other cells from reacting to, or even knowing about,

an event with no adverse consequences. In our HMG-centric

view, in mammalian cells histone H2B phosphorylation and

chromatin condensation might serve to prevent HMGB1

release. In general, chromatin modification might prevent

the release of anything that is abundant enough and soluble

enough to diffuse away as a signal, but can be soaked up by

modifying histones, DNA, or both.

It will be noticed that in this hypothesis the need for

chromatin modification relates to the need to broadcast a

molecular signal, or retain it: we see apoptosis and infor-

mation transmission as intimately linked. However, it takes

two to exchange information. Surviving cells are obviously

at the receiving end of such signalling in multicellular

organisms. But what about unicellular organisms, like

yeast? Yeast cells killed by oxygen radicals, antifungal

agents, or pheromones exhibit a phenotype that has been

described as apoptotic-like, and includes chromatin conden-

sation [28]. According to our hypothesis, chromatin is

modified to soak up a molecular signal: this requires that

such a signal exists, has a meaning, and indeed that a

receptor exists. Being unicellular does not eliminate the

need for communication: bacteria use ‘‘quorum sensing’’ to

gauge their density and proximity, and decide, for example

whether they might shift to forming a biofilm [29]. Yeasts

communicate with pheromones, and can shift to mycelial

habits [30]. In these proteomic days, it should not be

difficult to test whether there is a yeast protein that sticks

to the condensed chromatin of oxygen-killed cells, but not

to the chromatin of detergent-lysed cells. The reward for this

quest would be no less than a ‘‘death-code’’—the meaning

of death.

Acknowledgements

MEB is grateful to the high-altitude atmosphere (physical

and intellectual) of the Snowmass meeting on Chromatin

and Transcription. Conversations with Alessandra Agresti

and Eva Ugrinova provided useful insights. The authors’

laboratories are supported by grants from Associazione

Italiana Ricerca sul Cancro, CNR Programma Finalizzato

Biotecnologie, Ministero della Salute and Ministero dell’Is-

truzione, Universita e Ricerca Scientifica.

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