Chromatin conformation change code (4C) theory: A bio-system for gain of unprogramed and newcell function through irreversible creation ofchromatin structure plasticity with epigenetic
modifications via a lot of generation
メタデータ

言語: English
出版者:
公開日: 2020-06-21
キーワード (Ja):
キーワード (En): Chromatin conformation change code(4C) theory, Gain of unprogramed and new cellfunction, Irreversible creation of chromatin structure
plasticity, Epigenetic modifications; Lots ofgenerations, Immuno-electron microscopy study,
Pluri-potency, elasticity and flexibility of somatic cells
作成者: 中山, 建男, 中山, 雅美
メールアドレス:所属:

URL

http://hdl.handle.net/10458/5937

Chromatin conformation change code (4C) theory: A bio-system for gain of unprogramed and newcell function through irreversible creation of chromatin structure plasticity with epigenetic
modifications via a lot of generations**

Tatsuo Nakayama#, $, * and Masami Nakayama##

Section of Biochemistry and Molecular Biology, Department of Medical Sciences, Faculty of

Medicine, $Department of Life Science, Frontier Science Research Center, University of Miyazaki,
5200, Kihara, Kiyotake, Miyazaki, 889-1692, Japan*Corresponding author:

Tel.: +81 985 85 0872

E-mail: tnakayam@med.miyazaki-u.ac.jp (T. Nakayama)**The article is the modified version of Chapter 6 of the self-publishing monograph as follows:
Nakayama, T. and Nakayama, M.: Chromatin conformation change code (4C) theory: A bio-system
for gain of new cell function through irreversible creation of chromatin structure plasticity with
epigenetic modifications via a lot of generations. In: Chromatin Conformation Change Code (4C)
Theory on Gain of New Cell Function through Irreversible Creation of Chromatin Structure
Plasticity with Epigenetic Modifications via a Lot of Generations, Nakayama, T. and Nakayama, M.
(Eds.), Miyakonojoh-Insatsu, Inc. Miyazaki, Japan, pp. 167-201, 2015.

Available from two URLs

(http://opac2.lib.miyazaki-u.ac.jp/webopac/TC10123995 and/or http://hdl.handle.net/10458/5293).

1

Abstract
When higher eukaryotic cells firstly encounter moderate intra- and/or extra-cellular environmentchange, they newly acquire ability to cope with and/or overcome it through irreversible creation ofchromatin structure plasticity based on successive chromatin conformational change with epigenetic
modifications via a lot of generations.

Putative environment change recognition receptor/site(ECRR/ECRS) should recognize the environment change.

Putative chromatin conformation change

complex (4C) machinery should create irreversibly and separately varied chromatin structure plasticity ofproximal 5’-upstream regions of various transcription factor and/or chromatin-modifying enzyme genes
in individual cells of the same cell type.

We advance a chromatin conformation change code (4C)

theory for a bio-system to gain unprogramed and new cell function(s) through irreversible creation ofchromatin structure plasticity by the 4C machinery via a lot of generations, in order to adapt to theenvironment change recognized by ECRR/ECRS.

The 4C theory should be suitable as an explanation

for the manner of development and differentiation of higher eukaryotes.

Keywords
Chromatin conformation change code (4C) theory; Gain of unprogramed and new cell function;
Irreversible creation of chromatin structure plasticity; Epigenetic modifications; Lots of generations;
Immuno-electron microscopy study; Pluri-potency, elasticity and flexibility of somatic cells

Introduction
In eukaryotes, genome information and nuclear function should be mainly protected by nuclear
membrane acting as nucleus-cytoplasm barrier.

Additionally, communication and signal transduction

between nucleus and cytoplasm, both of which are essential for expressions of normal cell functions,should be preferentially carried out by the going and returning of large and/or small molecules throughnuclear pore acting as a guard station at the barrier.

By contrast, both of signal transduction on

unexpected and/or disadvantageous change in intra- and/or extra-cellular environment and transport ofuseless molecules to nucleus must be usually prevented by this barrier system.

Then, how higher

eukaryotic cells cope with and/or overcome unexpected and/or disadvantageous environment change,
when they firstly encounter it.

Following four typical countermeasures should be generally possible.

First, in the case of most severe change, cells should die because it is far ahead of their adaptation ability.
Second, in the case of considerable severe change, cells should cope with or overcome it by means ofalterations in genome information, such as point mutation, insertion, deletion, duplication andmultiplication on the DNA molecule via a lot of generations.
of species.

This mode should be a basis for evolution

Third, in the case of moderate change, cells should cope with or overcome it throughirreversible creation of chromatin structure plasticity caused by successive chromatin conformational2

(structural) changes with epigenetic modifications via a lot of generations.
for development and differentiation of cells.

This mode should be a basis

Fourth, in the case of minor change, cells should respondto it using only already acquired regulation mechanisms.
Since chemical modifications of histones with acetyl and methyl groups were first proposed toregulate RNA synthesis [1], the modulation of chromatin topology has been thought to be one of the mostfundamental and important events for expression of normal cell functions in eukaryotes.

Manners tomodulate chromatin structure with acetylation, methylation, phosphorylation, ubiquitination, sumoylation,
ADP ribosylation and so on have been intensively studied in a variety of life science fields.

Of these

epigenetic modifications, acetylation and deacetylation of particular Lys residues of core histones (H2A,
H2B, H3 and H4) catalyzed by chromatin-modifying enzymes, such as histone acetyltransferases (HATs)
and histone deacetylases (HDACs), are undoubtedly major ones.

For the last several decades, countless

numbers of researches on the acetylation and deacetylation (and also other epigenetic modifications) havebeen accumulated without interruption in more diverse life science fields; transcription/gene expression,
DNA replication, development, differentiation, memory, pluri-potency, clinical medicine and so on[2-28].
By gene targeting techniques in the chicken B cell line DT40 possessing homologous recombination
with a very high frequency [29, 30], we have systematically generated various mutants, all of which were
devoid of particular members of HDACs, HATs and transcription factors [31-45], besides those lacking
numerous members of histones and histone chaperones.

Systematic analyses of resultant DT40 mutants

HDAC2(-/-), Aiolos(-/-), Pax5(-), EBF1(-/-), Ikaros(-/-/+), Helios(-/-) and E2A(-/-) revealed theirinteresting participations in gene expressions of IgM H- and L-chains as follows.

In wild-type DT40

cells, HDAC2 as a supervisor regulates indirectly gene expressions of IgM H- and L-chains throughopposite control of those of Pax5, Aiolos and EBF1, and Ikaros plus E2A [31, 35].

Furthermore, in

HDAC2(-/-), IgM H- and L-chains are excessively accumulated based on their dramatically increased
gene expressions caused by drastic decreases in gene expressions of Pax5, Aiolos and EBF1, all of which
down-regulate gene expressions of the two immunoglobulin proteins [31, 33, 35].

On the other hand,

OBF1 is strongly suggested to up-regulate gene expressions of IgM H- and L-chains [46].
Very recently, we reported following remarkable and important phenomena by analyzing initiallygenerated HDAC2-deficient mutants [47], Pax5-deficient mutants [48] and secondly generated
HDAC2-deficient mutants [49], all of which were continuously cultivated for varying long periods.

In

individual clones of HDAC2(-/-) mutants, the accumulated IgM H- and L-chains at the early cultivation
stage are dramatically decreased attributed to their drastically decreased gene expressions in almost thesame changing pattern, caused by dramatic increases or decreases in gene expressions of Pax5, Aiolos
and EBF1 or OBF1 in distinct manners during cultivation (details will be discussed later) [49].

Theseresults obtained from wild-type DT40 cells and HDAC2(-/-) mutants at the early and late cultivation3

stages are schematically shown in Supplementary Figure S1.

Such diminutions of the accumulated IgM

H- and L-chains in all individual HDAC2(-/-) mutant clones during cultivation are really the third case of
the above-mentioned countermeasures.

Presumably, the manner for diminutions of IgM H- andL-chains accumulated in Pax5-deficient mutant cells Pax5(-) during cultivation moderately resembles thecase in HDAC2(-/-) mutant cells as a whole, although detailed molecular mechanisms to decrease gene
expressions of the two immunoglobulin proteins should be distinct between these two mutant cells [48,49].

In addition, acetylation levels of particular Lys residues of histone H3 (K9/H3, K14/H3, K18/H3,

K23/H3 and K27/H3) within chromatin surrounding proximal ~2.0 kb 5’-upstream regions of Pax5,
Aiolos, EBF1 and OBF1 genes are altered in distinct manners in all individual clones of HDAC2(-/-)
mutants during continuous cultivation (details will be discussed later) [50].
Here, based on these previously and newly obtained results from HDAC2(-/-) mutant cells, we
propose a universal concept, which we named the chromatin conformation change code (4C) theory, forthe bio-system to gain unprogramed and new cell function(s) through irreversible creation of chromatin
structure plasticity with epigenetic modifications via a lot of generations in higher eukaryotes.

Methods
Electron microscopy and immuno-electron microscopy
Electron microscopy and immuno-electron microscopy (using anti-chicken IgM H-chain antiserum as
primary antibody) were carried out on DT40 and initially generated HDAC2(-/-) mutant cells stocked at-80℃ [31, 35] in some different manners essentially as described [47-51].

For instance,

immuno-electron microscopy using pre-embedding staining procedure was done as follows.
Exponentially growing HDAC2(-/-) mutant cells at the early (E; ~10 days) and late (L; ~60 days)
cultivation stages and DT40 cells (W) were suspended in 1% bovine serum albumin (BSA) and collectedby a light centrifugation.

The resultant cell pellets were fixed with 4% paraformaldehyde in 0.1 M

phosphate buffer (PB) at 4℃ for 60 min.

After washing with PB the cell pellets were treated with orwithout 0.2% saponin in phosphate buffered saline (PBS) for 30 min to be permeable or non-permeable.
After washing with PBS the cell pellets were incubated with goat anti-chicken IgM H-chain antibody
A30-102A (Bethyl Laboratories Inc., TX, USA; diluted 1:1000 with 1% saponin in PBS) at 4℃ forovernight.

After washing with PBS the cell pellets were incubated with horseradish peroxidase(HRP)-labeled affinity-purified F(ab’)2 fragment Donkey anti-goat IgG(H+L) (Jackson ImmunoResearchLab. Inc., West Grove, PA, USA; diluted 1:1000 in PBS) at 4℃ for overnight.
the cell pellets were fixed with 1% glutaraldehyde in PBS at 4℃ for 5 min.

After washing with PBS

After washing with 50 mM

Tris-HCl buffer (pH 7.4) (TB) the cell pellets were incubated in 0.05% 3’, 3’-diaminobenzidine (DAB) in
TB for 20 min and complete DAB solution containing 0.005% H2O2 for 10 min.

The cell pellets were

post-fixed with 1% osmium tetroxide in TB containing 1% potassium ferrocyanide for 30 min,4

dehydrated with a series of ethanol and embedded in epoxy resin mixture.

Ultrathin sections were

contrasted with lead citrate for 20 sec and examined with a JEOL 1200 EX electron microscope (JEOL,Tokyo, Japan).
Immunocytochemistry using immuno-gold labeling was carried out as described [51].

Exponentiallygrowing DT40 (W) and HDAC2(-/-) mutant cells at the early (E) and late (L) cultivation stages werecollected by a light centrifugation.

The resultant cell pellets were fixed with 4% paraformaldehyde and0.1% glutaraldehyde in 0.1 M PB for 30 min.

After washing with PB the cells were post-fixed with 1%

osmium tetroxide in PB for 60 min, washed with PB, dehydrated in ethanol and embedded in Epon.
Ultrathin sections were picked up on 200-mesh gold grids coated with Formvar film and treated with 5%
sodium meta-periodate in distilled water for 30 min.

After rinsing in distilled water the sections weretreated with 5% normal horse serum (NHS) and 1% BSA in PBS for 10 min to block nonspecific binding
and incubated with goat anti-chicken IgM H-chain antibody A30-102A (diluted 1:1500 with 5% NHS, 1%
BSA in PBS) at room temperature for 60 min.

After rinsing in PBS the sections were incubated withbiotinylated horse anti-goat IgG (Vector Laboratories, Burlingame, CA, USA; diluted 1:200 with 1%
BSA in PBS) for 40 min.

After washing with PBS the sections were incubated with 8 nm colloidal gold

conjugated anti-biotin antibody (Vector Laboratories, Burlingame, CA, USA; diluted with 1% BSA in
PBS) for 30 min.

After washing with distilled water and drying the sections were contrasted with 2%

uranyl acetate in 70% methanol and Reynolds’ lead citrate and observed in a JEOL 1200EX transmission
electron microscope operating at 80 kV (JEOL, Tokyo, Japan).

Primary antibody was omitted or

replaced by normal goat serum as control.

Results
New findings and previous results [31, 35, 47-50] are as follows.

The HDAC2-deficiency in DT40

cells rapidly and dramatically increases mRNA (i.e., transcription/gene expression) levels of IgM H- andL-chains, followed by accumulation of the two immunoglobulin proteins probably within endoplasmic
reticulum (Figs. 1, 2 and 3, and Suppl. Figs. S2 and S3).

Surprisingly, excessively increased mRNAs

and proteins of IgM H- and L-chains at the early stage of cultivation are gradually decreased until the latecultivation stage in almost similar pattern in all examined individual clones (cl.2-1, cl.2-2, cl.2-3, cl.2-4,
cl.2-5 and cl.2-6) of HDAC2(-/-) mutants (Suppl. Figs. S4 and S5).

By contrast, remarkably, gene

expressions of various particular transcription factors and chromatin-modifying enzymes change indistinct patterns among these individual HDAC2(-/-) mutant clones (Suppl. Fig. S5).

To reduce theincreased protein levels of IgM H- and L-chains resulted from their increased gene expressions,apparently, following three distinct manners based on altered gene expressions of particular transcription
factors exist at the late cultivation stage in the six individual mutant clones (Suppl. Figs. S1(L) and S5)[49].

The manner in clone cl.2-1 seems to be dependent on OBF1 and different from that in DT40 cells.

5The manner in clones cl.2-2, cl.2-3, cl.2-4 and cl.2-5 seems to be dependent on Pax5 plus Aiolos andslightly similar to that in DT40 cells in appearance.

The manner in clone cl.2-6 seems to be dependent

on Pax5, Aiolos plus EBF1 and similar to that in DT40 cells in appearance.
To clarify these distinct manners, we performed neighboring overlapping tiling chromatin
immuno-precipitation (NotchIP or Notch-IP: this abbreviation also means IP on notch of chromatin)
assay on proximal ~2.0 kb 5’-upstream chromatin region (named as notch of chromatin) of each of theseparticular transcription factor genes (Suppl. Figs. S6, S7, S8 and S9) [50].

The results obtained by the

NotchIP assay revealed that such distinct manners should be fundamentally originated from irreversible
creation of varied chromatin structure plasticity surrounding proximal ~2.0 kb 5'-upstream regions ofcorresponding transcription factor genes with epigenetic modifications via a lot of generations during
cultivation.

To put it concretely, in wild-type DT40 cells having HDAC2 activity, chromatin structureof each of proximal ~2.0 kb 5'-upstream regions of genes encoding Pax5, Aiolos and EBF1 (which
down-regulate gene expressions of IgM H- and L-chains [35, 47, 49]) and OBF1 (which probably
up-regulates those of the two immunoglobulin proteins [46, 49]) is loose (open) form due to no binding
ability of histone H3 to DNA based on hyper- (high) acetylation levels of one or more of the particular
Lys residues (K9/H3, K14/H3, K18/H3, K23/H3 and K27/H3) [50].

Therefore, these four transcription

factor genes are transcribed at high levels in DT40 cells [35, 47, 49].

On the other hand, in all individualclones of HDAC2(-/-) mutants having no HDAC2 activity, chromatin structure of each of proximal ~2.0
kb 5’-upstream regions of these four factor genes is dramatically and severally altered based on varied
acetylation levels of one or more of the five particular Lys residues of histone H3 during cultivation,
resulting in alterations in their gene expression levels as follows [47, 49, 50].
In clone cl.2-1, at the early stage of cultivation, chromatin structure of each of proximal ~2.0 kb
5'-upstream regions of Pax5, Aiolos and EBF1 genes is tight (closed) form due to the binding ability ofhistone H3 to DNA based on hypo- (low or no) acetylation levels of one or more of the five particular Lys
residues (Suppl. Fig. S6), and as a result, transcriptions of these three genes are almost completely
suppressed to undetectable levels (Suppl. Fig. S5).

However, that of the OBF1 gene is somewhat loose

form due to the less binding ability of histone H3 to DNA based on slight (or considerably decreased)
hyper-acetylation levels, thereby its transcription is slightly decreased. By contrast, at the late cultivation
stage, chromatin structure of each of proximal 5'-upstream regions of Pax5, Aiolos and EBF1 genes
remains to be tight form due to the binding ability of histone H3 to DNA based on hypo-acetylationlevels; therefore, their transcriptions also remain unchanged at undetectable level.

On the other hand,chromatin structure of proximal ~2.0 kb 5'-upstream region of the OBF1 gene changes to tight form dueto the binding ability of histone H3 to DNA based on hypo-acetylation levels; therefore, its transcription
is suppressed to almost undetectable or very low level. These results supported the above-mentioned

6

inference (i.e., OBF1-dependent) on the manner of gene expressions of IgM H- and L-chains at the latecultivation stage in clone cl.2-1.
In clones cl.2-2 and cl.2-4, at the early stage of cultivation, as in clone cl.2-1, chromatin structure ofeach of proximal ~2.0 kb 5'-upstream regions of Pax5, Aiolos plus EBF1 genes or the OBF1 gene is tightor somewhat loose (or less tight) form based on hypo- or slight (or considerably decreased)
hyper-acetylation levels of one or more of the five particular Lys residues of histone H3 (Suppl. Figs. S7and S8). Therefore, transcriptions of the former three genes are almost completely suppressed and thatof the latter one gene is certainly decreased (Suppl. Fig. S5).

At the late cultivation stage, contrary tothis, chromatin structure of each of proximal ~2.0 kb 5'-upstream regions of Pax5, Aiolos and OBF1
genes changes to loose form based on hyper-acetylation levels, and their transcriptions are dramatically orcertainly increased. However, that of the EBF1 gene remains tight form based on hypo-acetylation levels,and its transcription remains low at almost undetectable level.

These results supported theabove-mentioned inference (i.e., Pax5- and Aiolos-dependent) on the manner of gene expressions of IgM
H- and L-chains at the late cultivation stage in clones cl.2-2 and cl.2-4 (and also cl.2-3 and cl.2-5).
Moreover, these four clones should be major type, since they resemble four initially generated
HDAC2(-/-) clones (cl.33-12, cl.33-28, cl.33-30 and cl.45-28) in several cellular characteristics [31, 35,
47, 49].
In clone cl.2-6, at the early stage of cultivation, as in clones cl.2-1, cl.2-2 and cl.2-4, chromatin
structure of each of proximal ~2.0 kb 5'-upstream regions of Pax5, Aiolos plus EBF1 genes or the OBF1
gene is tight or somewhat loose form based on hypo- or slight (or considerably decreased)
hyper-acetylation levels of one or more of the five particular Lys residues of histone H3 (Suppl. Fig.
S9). Therefore, transcriptions of the former three genes are almost completely suppressed and that of thelatter one gene is certainly decreased (Suppl. Fig. S5). By contrast, at the late cultivation stage,
chromatin structure of each of proximal ~2.0 kb 5'-upstream regions of Pax5, Aiolos, EBF1 and OBF1
genes changes to loose form based on hyper-acetylation levels, and their transcriptions are dramatically orcertainly increased.

These results supported the above-mentioned inference (i.e., Pax5-, Aiolos- andEBF1-dependent) on the manner of gene expressions of IgM H- and L-chains at the late cultivation stage
in clone cl.2-6.
Results on alterations in acetylation levels (hyper or hypo) of the five particular Lys residues ofhistone H3 (K9/H3, K14/H3, K18/H3, K23/H3 and K27/H3), form (loose or tight) of chromatin structureof proximal ~2.0 kb 5’-upstream regions of Pax5, Aiolos, EBF1 and OBF1 genes, and their mRNA
(transcription/gene expression) levels (high or low) during cultivation in individual HDAC2(-/-) mutant
clones cl.2-1, cl.2-2, cl.2-4 and cl.2-6 [49, 50] are roughly and schematically represented in
Supplementary Figure S10.

Concerning the above-stated results on the suppression of excessive gene

expressions of IgM H- and L-chains in HDAC2(-/-) mutants during cultivation, we should like to mention

7

some following important comments.

Excessively accumulated IgM H- and L-chains resulted from theirdramatically increased gene expressions in HDAC2(-/-) mutants just after their birth (by gene targeting
techniques) [31, 35, 47, 49] should be unfavorable and useless for them; therefore, the mutant cells havecome to acquire a new ability to diminish a large amount of the two immunoglobulin proteins as
uncomfortable environment change via a lot of generations during cultivation.

The manners to suppress

gene expressions of IgM H- and L-chains at the late cultivation stage in all of HDAC2(-/-) mutant clones,
even in clone cl.2-6 [49], should be surely distinct from the ordinary and reversible manner to regulate
their gene expressions in DT40 cells.

Because gene expressions of IgM H- and L-chains are indirectly

regulated by HDAC2 through opposite regulations of Pax5, Aiolos, EBF1, OBF1, and E2A gene
expressions in DT40 cells [35, 37].

In addition, gene expressions of various transcription factors andchromatin-modifying enzymes (i.e., Blimp1, PCAF, HDAC7, HDAC9 and others), besides Pax5, Aiolos,
EBF1 and OBF1, are dramatically and separately altered in individual HDAC2(-/-) mutant clones during
cultivation (Suppl. Fig. S5) [47, 49].

Therefore, remarkably, besides the alterations in gene expressionsof IgM H- and L-chains (and Pax5, Aiolos, EBF1 and OBF1) and in cell morphology, some otherunknown important cellular characteristics should be obviously altered among individual HDAC2(-/-)
mutant clones during cultivation.

Such presumable altered characteristics may be complicated anddiverse in individual clones of HDAC2(-/-) mutants, even though they are originally and completely thesame cell type.

Conclusions
Based on our morphological but insufficient findings, together with previous results mentioned above
and others [31, 33, 35, 37, 46-50], we slightly revised our previous hypothesis [50] and proposed an
all-inclusive hypothesis on manners for gain of unprogramed and new cell function to diminish artificiallyaccumulated IgM H- and L-chains through irreversible creation of varied chromatin structure plasticity ofseveral particular transcription factor genes with epigenetic modifications during continuous cultivation
in individual clones of HDAC2(-/-) mutants.
First, we assume putative environment change recognition receptor/site (ECRR/ECRS), which
participates in the recognition of accumulation of IgM H- and L-chains as unfavorable environmentchange (and probably acts in part in the signal transduction on the accumulation to chromatin structure)(Fig. 4).

In addition, putative chromatin conformation (structure) change complex (4C) machinery,

which is diverse and consists of a member of each of HATs plus HDACs and other factors, is supposed tocreate directly and irreversibly chromatin structure plasticity surrounding proximal 5'-upstream regions ofa set of particular genes (and also to act in part in the signal transduction) (Figs. 4 and 5).

Using

ECRR/ECRS, the 4C machinery and other components, the chain reaction of response to the environmentchange should occur as follows.

Large amounts of IgM H- and L-chains artificially caused by the8

HDAC2-deficiency (Suppl. Fig. S4) are first accumulated within endoplasmic reticulum of HDAC2(-/-)
mutant cells (Figs. 1, 2 and 3, and Suppl. Figs. S2 and S3).

Most of the accumulated immunoglobulin

proteins (which probably exist as a high molecular weight complex of IgM H- and L-chains each other[35]) are gradually secreted to outside of cells, whereas, as indicated by immuno-electron microscopy
some of them are transported to nuclear envelope but not inside of nucleus, and kept at peri-nuclear spaceat the early and also late cultivation stages (Figs. 1, 2, 3 and 4).

The accumulated immunoglobulin

proteins at peri-nuclear space should bind to ECRR/ECRS localized on inner nuclear membrane (where
hetero-chromatin is possibly located) (Fig. 4).

After ECRR/ECRS recognizes the accumulation of IgM

H- and L-chains as unfavorable environment change, the signal concerning it is genome-widelytransmitted to chromatin structure surrounding proximal 5’-upstream regions of numerous genes(probably located on several distinct chromosomes) encoding transcription factors, chromatin-modifying
enzymes, and related factors and enzymes.

Following the initial signal transduction, spontaneous

unbalanced response to the environment change is consecutively and separately converged not only on
the above-mentioned particular genes but also in individual clones of HDAC2(-/-) mutants.
In wild-type DT40 cells in which various members of HATs and HDACs are largely expressed [35,49], the 4C machinery for each of Pax5, Aiolos, EBF1 and OBF1 genes probably contains HDAC2 as
HDAC activity, a particular HAT member (e.g., GCN5) as HAT activity, and other factors (Fig. 5).

Onthe other hand, in all of individual clones of HDAC2(-/-) mutants, at the very early stage of cultivation(just soon after their birth by gene targeting techniques), bulk conformation of the 4C machinery should
be dramatically changed to remove or drastically reduce HAT activity (of the assumed member of HATs),
attributed to the HDAC2-deficiency.

Through the above-mentioned process and following continuous

cultivation, the 4C machinery should come to newly contain a different member of HDACs, a particular(same or different) member of HATs, and other factors, and thereby becomes varied.

The diversity ofalterations in chromatin structure is preferentially attributed to varied acetylation and deacetylation levelsof one or more of the particular Lys residues at N-terminal tail of histone H3 [50] caused by collaborationof proper member of each of HATs and HDACs in the protean 4C machinery.

These successive

epigenetic modifications with acetyl group of K9/H3 and K27/H3 (and also K14/H3, K18/H3 andK23/H3) lead to irreversible creation of distinct chromatin structure plasticity surrounding proximal
5'-upstream regions of the above-mentioned targeted genes (Suppl. Figs. S6, S7, S8, S9 and S10, and Fig.
6).

Accordingly, chromatin structure of proximal 5'-upstream regions possessing hyper-acetylationlevels of one or more of the particular Lys residues of histone H3 is loose form based on its no binding
ability to DNA, but that of proximal 5’-upstream regions possessing hypo-acetylation levels of one ormore of the particular Lys residues of histone H3 is tight form based on its binding ability to DNA (Fig.
6).

By contrast, probably, the 4C machinery cannot change chromatin structure surrounding open

reading frames (coding regions) of the targeted genes so much [50].

9Thus, as the need arises,transcription factor complex (TFC) machinery (which consists of RNA polymerase, proper transcription
factor(s), certain member of each of HATs and HDACs, and other factors) is able to bind to promoter
regions (or elements) within loose (but not tight) form of chromatin structure surrounding proximal
5’-upstream regions of the targeted (but not untargeted) genes (which have become latently active (butnot inactive) state), and thereby initiates their gene expressions (Figs. 5 and 6).

As a concrete result,individual HDAC2(-/-) mutant clones gain the same, unprogramed and new cell function to reduceincreased gene expressions of IgM H- and L-chains (resulting in their decreased protein levels) in almost
the same changing pattern through increased or decreased gene expressions of Pax5, Aiolos plus EBF1 or
OBF1 in distinct manners during simple continuous cultivation under the same conditions (Suppl. Fig.
S5) [35, 47, 49].

Naturally, such distinct manners are not under the control of HDAC2 but originallybased on irreversible creation of their distinct chromatin structure plasticity with epigenetic modifications
during continuous cultivation [50].

Remarkably, the six tested clones of HDAC2(-/-) mutants can beclearly classified into three different cell types exhibiting distinct functions, because they show three
dissimilar manners for gene expressions of transcription factors and chromatin-modifying enzymes (suchas Pax5, Aiolos, EBF1, OBF1, Blimp1, PCAF, HDAC9 and others) at the late cultivation stage (Suppl.
Figs. S1 and S5).

Besides, in one or more of these six mutant clones, as a whole, respective gene

expression patterns of PCAF, HDAC7, HDAC9, Ikaros and OBF1 are spontaneously and complicatedly
reversed in the midst of simple continuous cultivation, although those of Pax5, Aiolos, EBF1, E2A, PU.1
and Blimp1 are unchanged from the early to late cultivation stages.

Furthermore, there is a great

possibility that additional different cell types exist, if other individual mutant clones obtained [49] areanalyzed.

Consequently, individual clones of HDAC2(-/-) mutants acquire flexible, elastic andpluri-potential ability not only to adapt in distinct manners to an environment change but also to branch
off into diverse derivative cell types, which should exhibit varied characteristics (functions), even though
they are originally the same cell type and also established cell line.
We expanded the above-mentioned hypothesis on the exclusion of IgM H- and L-chains excessively
accumulated in HDAC2(-/-) mutants to a universal hypothetic concept on gain of unprogramed and newcell function(s) through irreversible creation of varied chromatin structure plasticity of a set of particulargenes with epigenetic modifications via a lot of generations (cell divisions) in higher eukaryotes.
When higher eukaryotic cells firstly in their life encounter the change in intra- and/or extra-cellularenvironment, in order to adapt to or eliminate the change (if uncomfortable), they gradually gain
unprogramed and new cell function(s) via a lot of generations.

Namely, the cells acquire the ability toadapt themselves to newly encountered environment change and/or to exclude the painful environmentchange.

Using ECRR/ECRS, the 4C machinery and other components, the cells should cause the chain

reaction of response to the environment change (Figs. 4 and 5).

First of all, the environment change

should be recognized by means of ECRR/ECRS, which may be localized nearby nuclear membrane as a10

nucleus-cytoplasm barrier (probably at inner nuclear membrane where hetero-chromatin is possibly
located) (Fig. 4).

Naturally, there is a possibility that putative particular molecule(s) should act asintermediary sensor at this step in the manner to recognize the environment change.

As a next step,

putative signal(s) concerning the change should be genome-widely transmitted to chromatin structure
within nucleus via a lot of generations (cell divisions).

The initial acceptance of the putative signal(s)may induce a slight alteration in chromatin structure of numerous chromatin-modifying enzymes,transcription factors, and related enzymes and factors with epigenetic modifications.

The transductionof the signal(s) and spontaneous unbalanced response to the environment change should be successively
repeated and converged into restricted chromatin structure surrounding proximal 5’-upstream regions of aset of particular members of the above-mentioned factor and enzyme genes.

Finally, this successive

signal transduction concerning the environment change should cause various epigenetic modifications ofhistones and/or DNA within the restricted chromatin structure of the aforesaid particular genes withacetyl group, methyl group, phosphate group and/or others.

Of these various epigenetic modifications,acetylation and deacetylation of particular Lys residues of core histones H2A, H2B, H3 and H4 may bemajor ones.
The 4C machinery, which consists of a particular member of each of HATs and HDACs, and otherfactors, should preferentially participate in acetylation and/or deacetylation among such epigenetic
modifications (Fig. 5).
should be diverse.

Participating positions of particular Lys residues and/or kinds of core histones

For instance, in the above-mentioned case [50], acetylation and/or deacetylation of

Lys-9, Lys-14, Lys-18, Lys-23 and Lys-27 residues of histone H3 are prominent.

Consequently,

epigenetic modifications of one or more of these Lys residues of histone H3 with acetyl group arechanged within chromatin structure surrounding proximal 5’-upstream regions of the above-mentioned
particular targeted genes via a lot of generations.

Distinct functions of the protean 4C machinery onsuch acetylation and/or deacetylation levels should be mainly based on different combinations of eachmember of HATs and HDACs as the components, because any HAT and HDAC members’ own activities
are probably unchangeable.

By contrast, the protean 4C machinery may not alter so much chromatin

structure surrounding open reading frames (coding regions) of corresponding genes.

The binding abilityof the N-terminal tail of histone H3 to DNA is tentatively and qualitatively deduced from acetylationlevels of one or more of these particular Lys residues, though which Lys residue(s) really and/or mainly
participates in the binding is still undefined.

Namely, hyper- (high) or hypo- (low or no) acetylationlevels should induce no binding or full binding ability, resulting in loose (open) or tight (closed) form ofchromatin structure (Fig. 5).

Thus, chromatin structure plasticity should be irreversibly created based on

successive conformation changes with epigenetic modifications.

These manners to create gradually andtardily chromatin structure plasticity for gain of unprogramed and new cell function(s) should be
obviously different from those to cause immediately and rapidly chromatin conformation change for gain11

of programed and ordinary cell function(s).
Whenever the need arises, the TFC machinery is able to bind to promoter regions (or elements) withinloose form of chromatin structure surrounding proximal 5'-upstream regions of targeted genes (which arelatently active state), followed by initiation of their gene expressions (Fig. 5).

By contrast, the TFC

machinery cannot bind to promoter regions (or elements) within tight form of chromatin structure
surrounding proximal 5’-upstream regions of untargeted genes (which are latently inactive state), andthereby cannot initiate their gene expressions.

Consequently, loose or tight form of chromatin structure

surrounding proximal 5’-upstream regions should respectively cause high or low (or no) transcription
levels of corresponding genes (Fig. 6).

Notably, there is a great possibility that manners for irreversible

creation of chromatin structure plasticity are distinct among individual cells of the same type, eventhough the environment change and signal(s) on it are the same for all of them.

That is, in order to gain

unprogramed and new cell function(s), individual cells each should possess ability not only to change
complicatedly and diversely chromatin structure surrounding proximal 5’-upstream regions of various
particular genes but also to alter separately chromatin structure of the same proximal 5’-upstream regionof a certain gene into varied forms.

Thus, gene expressions of the particular chromatin-modifying

enzymes and transcription factors should be changed diversely among individual cells via a lot ofgenerations, in spite of the same environment change.

In consequence, individual cells of the same typeare able to newly gain the same and/or distinct unprogramed cell function(s) in different manners in orderto accommodate themselves to a new environment.
In conclusion, for gain of unprogramed and new cell function(s), somatic cells (and even tumor cells)of higher eukaryotes are pluri-potential, elastic and flexible, all of which should be basically originatedfrom those of chromatin structure.

Namely, in order to adapt to intra- and/or extra-cellular environmentchange, individual somatic cells of higher eukaryotes possess ability to newly gain the same and/or
distinct unprogramed cell function(s) in different manners through irreversible creation of varied
chromatin structure plasticity with epigenetic modifications, i.e., from loose to tight forms or vice versaof chromatin structure surrounding proximal 5’-upstream regions of particular transcription factor andchromatin-modifying enzyme genes (Fig. 6).

Such loose or tight form of chromatin structure

surrounding the proximal 5’-upstream region should be latently active or inactive state for transcription of
the corresponding gene, although the proximal 5’-upstream region as mere nucleotide sequences may besilent state for expressions of most genome functions.

Variety of chromatin structure plasticity inindividual somatic cells should be triggered by the spontaneous unbalanced response to the environmentchange when they firstly encounter with it and then irreversibly accomplished by the successive
unbalanced convergence of the response via a lot of generations.

Naturally, different manners to create

varied chromatin structure plasticity in somatic cells should be certainly dependent on their antecedents.
Moreover, chromatin structure plasticity, regardless of whether its creation is in the course or was already12

completed, should be inherited to descendant generations associated with or without additional structuralchange via cell divisions.

Thus, remarkably, irreversible creation of chromatin structure plasticity

should occur in descendant cells but not in the cell which initially accepts the signal on the environmentchange, although reversible regulations of ordinary gene expressions and enzyme reactions occur in thecell itself which accepts proper signal (Fig. 7).

Probably, irreversible creation of chromatin structure

plasticity, with the intention of adapting to the environment change, should occur inevitably but notincidentally and/or neutrally.
The proximal 5’-upstream chromatin region (as loose or tight form) should direct the switch (on oroff) for latent transcription ability of the corresponding gene through irreversible creation of chromatin
structure plasticity; therefore, besides “notch” from a structural side-view as mentioned above, the5’-upstream chromatin region could be regarded as “director” from a functional side-view.

Naturally,

the notch (or director) covers specific nucleotide sequences of transcriptional elements (such as promoter,
operator and others) and also their neighboring nucleotide sequences.

Remarkably, the real recipient of

the signal on the environment change may be just chromatin structure possessing self-reformation ability
itself as three-dimensional conformation which is dynamic and changeable between loose and tight forms,
but not mere chromatin and chromosome themselves as one- (or two-) dimensional conformation which
are static and unchangeable.

That is, the chromatin structure of proximal 5’-upstream region (notch or

director), as dynamic and changeable three-dimensional conformation, possesses two fundamental
abilities, i.e., to receive the signal concerning intra- and/or extra-cellular environment change and todirect the switch (on or off) for latent transcription ability of the corresponding gene through its
irreversible chromatin conformation change responsive to the signal, although concrete data are notenough to support these ideas so far.

In consequence of these complicated biological events, higher

eukaryotes acquire pluri-potential, elastic and flexible ability to create diverse derivative cell types
possessing varied characteristics (functions) in distinct manners from the same type of somatic cells, inorder to accommodate themselves to new intra- and/or extra-cellular environment.

Naturally, in somecases, even neighboring cells derived from the same cell type are probably regarded as extra-cellularenvironment each other.

Moreover, in higher eukaryotes diverse kinds of somatic cells should share andexpress cooperatively vital functions each other.
Finally, we name such bio-system for gain of unprogramed and new cell function(s) throughirreversible creation of chromatin structure plasticity with epigenetic modifications, which should be oneof the most fundamental and important manners for life conservation and cell type determination ofhigher eukaryotes, a chromatin conformation (structure) change code (4C) theory.

Probably, thesupposed number of codes in the 4C theory, which should determine complicated and variedcharacteristics (functions) of higher eukaryotic cells, can be roughly estimated based on combination(multiplication) of the number of candidate genes and that of codes for each of these genes as follows.

13

Most influential candidates should be genes of transcription factors, chromatin-modifying enzymes, andrelated factors and enzymes, which are necessary for gain of varied cell functions and specificities ofhigher eukaryotes.

The number of codes for each of these candidate genes should be two.

Becauseloose or tight form of proximal 5’-upstream chromatin region, as latently active or inactive state, directs
the switch (on or off) for transcription ability of the corresponding gene.

Therefore, the 4C theory

should open the door for acquisition of unprogramed and new cell function(s) of higher eukaryotes andinnovate the general notion on nature of somatic cells.

Naturally, programed, complicated and diverse

cell functions are orderly and systematically gained throughout their development and differentiation.
Nevertheless, the 4C theory should be suitable as an explanation for the development and differentiationof higher eukaryotes, because action of putative signal(s) concerning environment changes seems to fairly
resemble that of certain players (such as hormone, cytokine, nerve-transmission substance, etc.), which
participate in cell-cell, tissue-tissue and/or organ-organ interactions (communications) throughout these
two fundamental life phenomena.
There are several crucial questions to be clarified in the 4C theory on exclusion of excessive IgM Hand L-chains artificially accumulated in HDAC2(-/-) DT40 mutant cells [47, 49, 50].

1) Despite the

HDAC2-deficiency, why acetylation levels of one or more of K9, K14, K18, K23 and K27 residues ofhistone H3 within chromatin structure (of ~10 nucleosomes) surrounding proximal 5’-upstream regions of
Pax5, Aiolos and EBF1 genes are decreased at the early stage of cultivation in HDAC2(-/-) mutants.

2)

Why the decreased acetylation levels of one or more of the particular Lys residues of histone H3 withinproximal 5’-upstream chromatin regions of these three genes are increased during cultivation.
case of the OBF1 gene is reverse.

Why the3) It must be determined which Lys residue(s) of K9, K14, K18, K23

and K27 of histone H3 is really and/or mainly involved in its binding to DNA within proximal
5’-upstream chromatin regions of Pax5, Aiolos, EBF1 and OBF1 genes.

4) Functional and stericdifferences between loose and tight forms (based on hyper- and hypo-acetylation levels of one or more of
the particular Lys residues of histone H3) of chromatin structure surrounding proximal 5’-upstream
regions of these four genes must be clarified more minutely.

5) Why changing patterns in acetylationlevels of the particular Lys residues of histone H3 during cultivation in individual transcription factor
genes differ among individual mutant clones.

6) Why changing patterns in acetylation levels of theparticular Lys residues of histone H3 during cultivation in each individual transcription factor gene differ
within individual mutant clones.

7) How the 4C machinery (which acts in irreversible creation ofchromatin structure plasticity of proximal 5’-upstream region) differs from the well-known
chromatin-modifying machinery, although the TFC machinery (which acts in transcription of open
reading frame of the corresponding gene) may be the same as the well-known transcription machinery.
8) To demonstrate the 4C theory, both of putative ECRR/ECRS as a first player to recognize theenvironment change and putative 4C machinery as a final player to irreversibly create chromatin structure

14plasticity must be clarified.

9) As concrete approach to generalize the 4C theory, for instance,

elucidation of influences of changes in temperature, atmosphere and/or nutrition on ability to gain
unprogramed and new cell function(s) in established cell lines via a lot of generations and in model
animals (such as C. elegans, Drosophila, Xenopus, mice, rats and others) during development anddifferentiation should be very effective and powerful.

Acknowledgments
The authors are grateful to Drs. T. Suganuma and A. Sawaguchi for electron microscopy andimmuno-electron microscopy study.

And the authors thank Dr. K. Toshimori for experimental support.

References[1] Allfrey, V., Faulker, R. M. and Mirsky, A. E.: Acetylation and methylation of histones and their
possible role in the regulation of RNA synthesis. Proc. Natl. Acad. Sci. USA, 51, 786-794, 1964.
[2] Brownell, J. E., Zhou, J., Rannali, T., Kobayashi, R., Edmondson, D. G., Roth, S. Y. and Allis, C. D.:
Tetrahymena histone acetyltransferase A: a homolog to yeast Gcn5 linking histone acetylation to geneactivation. Cell 84, 843-851, 1996.
[3] Ogryzko, V. V., Schiltz, R. L., Russanova, V., Howard, B. H. and Nakatani, Y.: The transcriptional
coactivators p300 and CPB are histone acetyltransferases. Cell 87, 953-959, 1996.
[4] Taunton, J., Hassig, C. A. and Schreiber, S. L.: A mammalian histone deacetylase related to the yeasttranscriptional regulator Rpd3p. Science 272, 408-411, 1996.
[5] Brown, C. E., Lechner, T., Howe, L. and Workman, J. L.: The many HATs of transcription
coactivators. Trends Biochem. Sci. 25, 15-19, 2000.
[6] Cheung, W. L., Briggs, S. D. and Allis, C. D.: Acetylation and chromosomal functions. Curr. Opin.
Cell Biol. 12, 326-333, 2000.
[7] Turner, B. M.: Histone acetylation and an epigenetic code. Bioessays 22, 836-845, 2000.
[8] Roth, S. Y. Denu, J. M. and Allis, C. D.: Histone acetyltransferases. Annu. Rev. Biochem. 70, 81-120,
2001.
[9] Carrozza, M. J., Utley, R. T., Workman, J. L. and Cote, J.: The diverse functions of histone
acetyltransferase complexes. Trend. Genet. 19, 321-329, 2003.
[10] Yang, X. J. and Seto, E.: Collaborative spirit of histone deacetylases in regulating chromatin
structure and gene expression. Curr. Opin. Genet. Dev. 13, 143-153, 2003.
[11] Margueron, R., Trojer, P. and Reinberg, D.: The key to development: interpreting the histone code?
Curr. Opin. Genet. Dev. 15, 163-176, 2005.
[12] Goldberg, A. D., Allis, C. D. and Bernstein, B. E.: Epigenetics: a landscape takes shape. Cell 128,635-638, 2007.

15

[13] Shahbazian, M. D. and Grunstein, M.: Functions of site-specific histone acetylation anddeacetylation. Annu. Rev. Biochem. 76, 75-100, 2007.
[14] Allis, C. D., Berger, S. L., Cote, J., Dent, S., Jenuwien, T., Kouzarides, T., Pillus, L., Reinberg, D.,
Shi, Y., Shiekhattar, R., Shilatifard, A., Workman, J. and Zhang, Y.: New nomenclature forchromatin-modifying enzymes. Cell 131, 633-636, 2007.
[15] Lee, K. K. and Workman, J. L.: Histone acetyltransferase complexes: one size doesn’t fit all. Nat.
Rev. Mol. Cell Biol. 8, 284-295, 2007.
[16] Berger, S. L.: The complex language of chromatin regulation during transcription. Nature 447,
407-412, 2007.
[17] Suganuma, T. and Workman, J. L.: Crosstalk among histone modifications. Cell 135, 604-607, 2008.
[18] Kohn, K. W., Aladjem, M. I., Weinstein, J. N. and Pommier, Y.: Chromatin challenges during DNA
replication: A systems representation. Mol. Biol. Cell 19, 1-7, 2008.
[19] Selvi, R. B. and Kundu, T. K.: Reversible acetylation of chromatin: implication in regulation of gene
expression, disease and therapeutics. Biotech. J. 4, 375-390, 2009.
[20] Javierre, B. M., Hemando, H. and Ballestar, E.: Environmental triggers and epigenetic deregulation
in autoimmune disease. Discov. Med. 12, 535-545, 2011.
[21] Bannister, A. J. and Kouzarides, T.: Regulation of chromatin by histone modifications. Cell Res. 21,
381-395, 2011.
[22] Verrier, L., Vandromme, M. and Trouche, D.: Histone demethylases in chromatin cross-talks. Biol.
Cell 103, 381-401, 2011.
[23] Butler, J. S., Koutelou, E., Schibler, A. C. and Dent, S. Y.: Histone-modifying enzymes: regulatorsof developmental decisions and drivers of human disease. Epigenomics 4, 163-177, 2012.
[24] Kooistra, S. M. and Helin, K.: Molecular mechanisms and potential functions of histone deacetylases.
Nat. Rev. Mol. Cell. Biol. 13, 297-311, 2012.
[25] Graff, J. and Tsai, L.-H.: Histone acetylation: molecular mnemonics on the chromatin. Nat. Rev.
Neurosci. 14, 97-111, 2013.
[26] Chen, T. and Dent, S. Y. R.: Chromatin modifiers and remodellers: regulators of cellulardifferentiation. Nat. Rev. Genet. 15, 93-106, 2014.
[27] T. W.-W. and Reinberg, D.: Chromatin futures and the epigenetic regulation of pluripotency states in
ESCs. Development 141, 2376-2390, 2014.
[28] Morgan, M. A. and Shilatifard, A.: Chromatin signatures of cancer. Genes and Dev. 29, 238-249,
2015.
[29] Baba, T. W., Giroir, B. P. and Humphries, E. H.: Cell lines derived from avian lymphomas exhibit
two distinct phenotypes. Virology 144, 139-151, 1985.

16

[30] Buerstedde, J.-M. and Takeda, S.: Increased ratio of targeted to random integration after transfectionof chicken B cell lines. Cell 67, 179-188, 1991.
[31] Takami, Y., Kikuchi, H. and Nakayama, T.: Chicken histone deacetylase-2 controls the amount of
the IgM H-chain at the steps of both transcription of its gene and alternative processing of its pre-mRNAin the DT40 cell line. J. Biol. Chem. 274, 23977-23990, 1999.
[32] Takami, Y. and Nakayama, T.: N-terminal region, C-terminal region, nuclear export signal anddeacetylation activity of histone deacetylase-3 are essential for the viability of the DT40 chicken B cellline. J. Biol. Chem. 275, 16191-16201, 2000.
[33] Takechi, S., Adachi, M. and Nakayama, T.: Chicken HDAC2 down-regulates IgM light chain genepromoter activity. Biochem. Biophys. Res. Commun. 299, 263-267, 2002.
[34] Kikuchi, H., Takami, Y. and Nakayama, T.: GCN5: a supervisor in all-inclusive control of vertebratecell cycle progression through transcription regulation of various cell cycle-related genes. Gene 347,
83-97, 2005.
[35] Nakayama, M., Suzuki, H., Yamamoto-Nagamatsu, N., Barman, H. K., Kikuchi, H., Takami, Y.,
Toyonaga, K., Yamashita, K. and Nakayama, T.: HDAC2 controls IgM H and L-chain gene expressions
via EBF1, Pax5, Ikaros, Aiolos and E2A gene expressions. Genes Cells 12, 359-373, 2007.
[36] Kikuchi, H. and Nakayama, T.: GCN5 and BCR signaling collaborate to induce pre-mature B cell
apoptosis through depletion of ICAD and IAP2 and activation of caspase activities. Gene 419, 48-55,
2008.
[37] Kikuchi, H., Barman, H. K., Nakayama, M., Takami, Y. and Nakayama, T.: Studies on epigenetic
control of B cell functions using the DT40 cell line. Advances in Genetics Research 2, Urbano K. V.
(Ed.), Nova Science Publishers, Inc. NY, pp153-166, 2010.
[38] Kikuchi, H., Kuribayashi, F., Takami, Y., Imajoh-Ohmi, S. and Nakayama, T.: GCN5 regulates theactivation of PI3K/Akt survival pathway in B cells exposed to oxidative stress via controlling gene
expressions of Syk and Btk. Biochem. Biophys. Res. Commun. 405, 657-661, 2011.
[39] Kikuchi, H., Kuribayashi, F., Kiwaki, N., Takami, Y. and Nakayama, T.: GCN5 regulates thesuperoxide-generating system in leukocytes via controlling gp91-phox gene expression. J. Immunol. 186,3015-3022, 2011.
[40] Kikuchi, H., Kuribayashi, F., Imajoh-Ohmi, S., Nishitoh, Y., Takami, Y. and Nakayama, T.: GCN5
protects vertebrate cells against UV-irradiation via controlling gene expression of DNA polymerase η. J.
Biol. Chem. 287, 39842-39849, 2012.
[41] Kikuchi, H., Nakayama, M., Kuribayashi, F., Imajoh-Ohmi, S., Nishitoh, H., Takami, Y. andNakayama T.: GCN5 is essential for IRF-4 gene expression followed by transcriptional activation of
Blimp-1 in immature B cells. J. Leukoc. Biol. 95, 399-404, 2014.

17

[42] Kikuchi, H., Nakayama, M., Kuribayashi, F., Imajoh-Ohmi, S., Nishitoh, H., Takami, Y. andNakayama T.: Protein kinase Cθ is oppositely regulated by GCN5 and RBF1 in immature B cells. FEBS
Lett. 588, 1739-1742, 2014.
[43] Kikuchi, H., Nakayama, M., Kuribayashi, F., Imajoh-Ohmi, S., Nishitoh, H., Takami, Y. andNakayama T.: GCN5 is involved in regulation of immunoglobulin heavy chain gene expression in
immature B cells. Gene 544, 19-24, 2014.
[44] Kikuchi, H., Nakayama, M., Kawai, C., Kuribayashi, F., Mimuro, H., Imajoh-Ohmi, S., Nishitoh, H.,
Takami, Y. and Nakayama T.: The histone acetyltransferase p300/CBP-associated factor acts as aneffective suppressor of secretory immunoglobulin synthesis in immature cells. Microbiol. Immunol. 59,
243-247, 2015.
[45] Kikuchi, H., Kuribayashi, F., Mimuro, H., Imajoh-Ohmi, S., Nakayama, M., Takami, Y., Nishitoh, H.
and Nakayama, T.: Lack of GCN5 remarkably enhances the resistance against prolonged endoplasmic
reticulum stress-induced apoptosis through up-regulation of Bcl-2 gene expression. Biochem. Biophys.
Res. Commun. 463, 870-875, 2015.
[46] Takechi, S., Adachi, M. and Nakayama, T.: Cloning and characterization of the chick Oct binding
factor OBF-1. Biochim. Biophysica Acta 1577, 466-470, 2002.
[47] Nakayama, M. and Nakayama, T.: Protein and mRNA levels of IgM H- and L-chains artificially andexcessively accumulated in HDAC2-deficient DT40 mutants are gradually reduced via a lot ofgenerations during continuous cultivations. In: Chromatin Conformation Change Code (4C) Theory on
Gain of New Cell Function through Irreversible Creation of Chromatin Structure Plasticity with
Epigenetic Modifications via a Lot of Generations, Nakayama, T. and Nakayama, M. (Eds.),
Miyakonojoh-Insatsu, Inc. Miyazaki, Japan, pp. 11-44, 2015.

Available from two URLs

(http://opac2.lib.miyazaki-u.ac.jp/webopac/TC10123995 and/or http://hdl.handle.net/10458/5293).

The

article with a slight modification is in preparation for publication.
[48] Nakayama, M. and Nakayama, T.: Generation of Pax5-deficient DT40 mutants, Pax5(-), and protein
and mRNA levels of artificially accumulated IgM H- and L-chains in Pax5(-) are rapidly reduced via a lotof generations during continuous cultivation. In: Chromatin Conformation Change Code (4C) Theory on
Gain of New Cell Function through Irreversible Creation of Chromatin Structure Plasticity with
Epigenetic Modifications via a Lot of Generations, Nakayama, T. and Nakayama, M. (Eds.),
Miyakonojoh-Insatsu, Inc. Miyazaki, Japan, pp. 45-70, 2015.

Available from two URLs

(http://opac2.lib.miyazaki-u.ac.jp/webopac/TC10123995 and/or http://hdl.handle.net/10458/5293).

The

article with a slight modification is in preparation for publication.
[49] Nakayama, M. and Nakayama, T.: Artificially and excessively accumulated IgM H- and L-chains in
HDAC2(-/-) DT40 mutants are gradually and dramatically reduced in distinct manners in individual
mutant clones via a lot of generations during continuous cultivation. In: Chromatin Conformation Change18

Code (4C) Theory on Gain of New Cell Function through Irreversible Creation of Chromatin Structure
Plasticity with Epigenetic Modifications via a Lot of Generations, Nakayama, T. and Nakayama, M.
(Eds.), Miyakonojoh-Insatsu, Inc. Miyazaki, Japan, pp. 71-103, 2015.

Available from two URLs

(http://opac2.lib.miyazaki-u.ac.jp/webopac/TC10123995 and/or http://hdl.handle.net/10458/5293).

The

article with a slight modification is in preparation for publication.
[50] Nakayama, M. and Nakayama, T.: A fundamental manner for irreversible creation of chromatin
structure plasticity with epigenetic modifications for gain of new cell function to exclude accumulated
IgM H- and L-chains in HDAC2(-/-) DT40 mutants via a lot of generations during continuous cultivation.
In: Chromatin Conformation Change Code (4C) Theory on Gain of New Cell Function throughIrreversible Creation of Chromatin Structure Plasticity with Epigenetic Modifications via a Lot ofGenerations, Nakayama, T. and Nakayama, M. (Eds.), Miyakonojoh-Insatsu, Inc. Miyazaki, Japan, pp.
104-166, 2015.

Available from two URLs (http://opac2.lib.miyazaki-u.ac.jp/webopac/TC10123995

and/or http://hdl.handle.net/10458/5293).

The article with a slight modification is in preparation for

publication.
[51] Bendayan, M. and Zollinger, M.: Ultrastructural localization of antigenic sites on osmium-fixedtissues applying the protein A-gold technique. J. Histochem. Cytochem. 31, 101-109, 1983.

19

Figure legends
Figure 1.

Localization of IgM H-chain at peri-nuclear space, endoplasmic reticulum and cellsurface of HDAC2(-/-) DT40 mutants
HDAC2(-/-) mutant cells were collected at the early stage of cultivation.
microscopy using anti-chicken IgM H-chain antiserum was carried out.
microscopy after treatment without saponin.
saponin.

Immuno-electronA) Immuno-electronB) ~ E) Immuno-electron microscopy after treatment with

Arrows P, E and S indicate positive signals of IgM H-chain proteins localized at peri-nuclear

space, endoplasmic reticulum and surface, respectively.

Figure 2.

Localization of IgM H-chain at peri-nuclear space of HDAC2(-/-) DT40 mutants

HDAC2(-/-) mutant cells were collected at the early stage of cultivation, and immuno-electron
microscopy using anti-chicken IgM H-chain antiserum was carried out.
microscopy of two individual single mutant cells.
arrows in A) and B), respectively.

A) and B) Immuno-electronD) and E) Enlarged versions of parts indicated byC) and F) Enlarged versions of immuno-electron microscopy of

peri-nuclear space of one DT40 cell and another HDAC2(-/-) mutant cell, respectively.

Accumulated

IgM H-chain proteins were observed in peri-nuclear space (indicated by arrows) of all HDAC2(-/-)
mutant cells.

Figure 3.

Localization of IgM H-chain at peri-nuclear space of HDAC2(-/-) DT40 mutants at earlyand late stages of cultivation
HDAC2(-/-) mutant cells were collected at the early (E) and late (L) stages of cultivation, andimmuno-electron microscopy using anti-chicken IgM H-chain antiserum was carried out.
Immuno-electron microscopy of one HDAC2(-/-) mutant cell at the early cultivation stage.
Enlarged versions of parts indicated by arrows b and c in A).

B) and C)D), G) and J) Immuno-electron

microscopy of three independent HDAC2(-/-) mutant cells at the late cultivation stage.
K) Enlarged versions of parts indicated by arrows e, f, h, i and k in D), G) and J).
was identical with that of Ref. 47.

A)E), F), H), I) and

The mutant cell in G)

Accumulated IgM H-chain proteins were observed in peri-nuclear

space of all HDAC2(-/-) mutant cells at the early and late cultivation stages.

Figure 4.

Localization of IgM H-chain at peri-nuclear space, and a model for signal transduction

on accumulated IgM H- and L-chains to chromatin structure in HDAC2(-/-) DT40 mutants
Left panel: A portion of peri-nuclear space (where IgM H-chain proteins were accumulated) of the
HDAC2(-/-) mutant cell (indicated by an arrow P at lower position in Figure 1C) was reversely enlarged.
Right panel: Signal concerning artificially accumulated IgM H- and L-chains in peri-nuclear space of the
HDAC2(-/-) mutant cell was repeatedly transmitted to chromatin structure, followed by unbalanced

20response for the signal and its convergence to various particular genes in individual mutant clones.
ECRR/ECRS: environment change recognition receptor/site.

4C machinery: chromatin conformation

change complex (4C) machinery.

Figure 5.

Summary on alterations in mRNA levels, acetylation levels of particular Lys residues ofhistone H3, and chromatin structure of proximal 5’-upstream regions of Pax5, Aiolos, EBF1 andOBF1 genes in individual clones of HDAC2(-/-) DT40 mutants during cultivation
Alterations in mRNA (gene expression) levels (-, ++ or ++++), acetylation levels (Ac; -, ++ or ++++)of particular Lys residues of histone H3 and chromatin structure (form; loose or tight) of proximal
5’-upstream regions of Pax5, Aiolos, EBF1 and OBF1 genes in HDAC2(-/-) mutant clones (cl.2-1, cl.2-2,
cl.2-4 and cl.2-6) at the early (E) and late (L) stages of cultivation and DT40 cells (W) are schematically
represented.

Chromatin conformation change complex (4C) machinery should generally containparticular member of each of HATs plus HDACs and other factors.

Transcription factor complex (TFC)machinery should generally contain RNA polymerase (RPase), particular transcription factors and others.

Figure 6.

Chromatin conformation change code (4C) theory: A model for gain of unprogramed

and new cell function through irreversible creation of chromatin structure plasticity with epigenetic
modifications via a lot of generations
Irreversible creation of chromatin structure plasticity with epigenetic modifications occurs withinproximal 5’-upstream region (notch of chromatin) but not within open reading frame of particular gene
via a lot of generations.

Tight or loose form of chromatin structure is based on hypo- or

hyper-acetylation levels of particular Lys residues of histone H3, and causes low or high transcription(gene expression) levels.

Figure 7.

Irreversible creation of chromatin structure plasticity surrounding proximal

5’-upstream region with epigenetic modifications in descendent cells, and reversible regulations ofordinary gene expression and enzyme reaction in cells that initially accept proper signal
Upper panel: Irreversible creation of chromatin structure plasticity surrounding proximal 5’-upstream
region of particular gene with epigenetic modifications occurs in descendant cells but not in initial cellthat accepts environment change signal.

Ac, Ac/2 and Ac/10 indicate qualitatively hyper-, considerable

hyper- and somewhat hyper-acetylation levels of particular Lys residues of core histones (e.g., histoneH3), respectively.

Middle and lower panels: Regulations of both gene expression (on transcriptional

regulatory elements) and enzyme reaction occur reversibly in the cell itself, which accepts proper signal.
Ac and P indicate acetylation, phosphorylation and/or other chemical modifications, respectively.

21

Supplementary Figure S1.

Manners for control of gene expressions of IgM H- and L-chainsthrough control of gene expressions of particular transcription factors in the presence or absence of
HDAC2 in wild-type DT40 cells (W) or all and individual clones of HDAC2(-/-) DT40 mutants atearly (E) and late (L) stages of cultivation
The figure is a set of Figures 4-8, 4-9 and 4-10 of Ref. 49.

Supplementary Figure S2.

Localization of IgM H-chain in wild-type DT40 cells and HDAC2(-/-)

DT40 mutants
Upper panel: Immuno-electron microscopy using anti-chicken IgM H-chain antiserum was carried out
on wild-type DT40 cells (left) and HDAC2(-/-) mutants at the early cultivation stage (center and right).
Lower panel: Enlarged versions of DT40 and HDAC2(-/-) mutant cells indicated by arrows in the upper
panel.

Positive signals of IgM H-chains are observed both at cell surface and cytoplasm of HDAC2(-/-)

mutant cells but only at cell surface of DT40 cells.

Supplementary Figure S3.

Localization of dense fraction due to accumulated IgM H-chain in

HDAC2(-/-) DT40 mutants
Electron microscopy was carried out on DT40 (upper panel; left and center) and HDAC2(-/-) mutant
cells at the early cultivation stage (lower panel; left and center).

Enlarged versions of DT40 and

HDAC2(-/-) mutant cells indicated by arrows in the centers are shown in the right of upper and lower
panels, respectively.

Dense fractions due to accumulated IgM H-chains are observed only in

HDAC2(-/-) mutant cells.

Supplementary Figure S4.

Alterations in protein levels of IgM H- and L-chains in individualclones of HDAC2(-/-) DT40 mutants during cultivation
The figure is identical with Figure 4-2 of Ref. 49.

Supplementary Figure S5.

Alterations in gene expressions of IgM H- and L-chains, members of

HATs, HDACs and transcription factors in individual clones of HDAC2(-/-) DT40 mutants during
cultivation
The figure is identical with Figure 4-6 of Ref. 49 and shown with some modifications.

Supplementary Figure S6.

Alterations in acetylation levels of particular Lys residues of histone

H3 within proximal 5’-upstream chromatin regions of Pax5, Aiolos, EBF1 and OBF1 genes in clone
cl.2-1 of HDAC2(-/-) DT40 mutants during cultivation
The figure is a set of Figures 5-1, 5-5, 5-9 and 5-13 of Ref. 50.

22

Supplementary Figure S7.

Alterations in acetylation levels of particular Lys residues of histone

H3 within proximal 5’-upstream chromatin regions of Pax5, Aiolos, EBF1 and OBF1 genes in clone
cl.2-2 of HDAC2(-/-) DT40 mutants during cultivation
The figure is a set of Figures 5-2, 5-6, 5-10 and 5-14 of Ref. 50.

Supplementary Figure S8.

Alterations in acetylation levels of particular Lys residues of histone

H3 within proximal 5’-upstream chromatin regions of Pax5, Aiolos, EBF1 and OBF1 genes in clone
cl.2-4 of HDAC2(-/-) DT40 mutants during cultivation
The figure is a set of Figures 5-3, 5-7, 5-11 and 5-15 of Ref. 50.

Supplementary Figure S9.

Alterations in acetylation levels of particular Lys residues of histone

H3 within proximal 5’-upstream chromatin regions of Pax5, Aiolos, EBF1 and OBF1 genes in clone
cl.2-6 of HDAC2(-/-) DT40 mutants during cultivation
The figure is a set of Figures 5-4, 5-8, 5-12 and 5-16 of Ref. 50.

Supplementary Figure S10.

Summary on alterations in acetylation levels (hyper or hypo) ofparticular Lys residues of histone H3, chromatin structure (loose or tight) of proximal 5’-upstream
regions of Pax5, Aiolos, EBF1 and OBF1 genes, and their mRNA (gene expression) levels (high orlow) in DT40 cells (W) and clones cl.2-1, cl.2-2, cl.2-4 and cl.2-6 of HDAC2(-/-) DT40 mutants atearly (E), middle (M) and late (L) stages of cultivation
The figure is identical with Figure 5-21 of Ref. 50 and shown with some modifications.

23