Epigenetics and Regeneration

Nobuyasu Maki and Hironobu Kimura

Abstract During newt lens regeneration a unique transdifferentiation event
occurs. In this process, dorsal iris pigmented epithelial cells transdifferentiate into
lens cells. This system should provide a new insight into cellular plasticity in basic
and applied research. Recently, a series of approaches to study epigenetic repro-
gramming during transdifferentiation have been performed. In this review, we
introduce the regulation of dynamic regulation of core-histone modifications and
the emergence of an oocyte-type linker histone during transdifferentiation. Finally,
we show supporting evidence that there are common strategies of reprogramming
between newt somatic cell in transdifferentiation and oocytes after somatic cell
nuclear transfer.


1 Introduction........................................................................................................................ 238
2 Newt Lens Transdifferentiation ........................................................................................ 238
2.1 A Key Biological Event ........................................................................................... 238
2.2 Structural Changes in the Nucleus........................................................................... 240
2.3 Gene Expression ....................................................................................................... 240
3 Epigenetics in Newt Lens Transdifferentiation................................................................ 241
3.1 Core Histone Modifications ..................................................................................... 241

N. Maki (&)  H. Kimura
Institute of Protein Research,
Osaka University, 3-2 Yamadaoka, Suita-Shi, Osaka 565-0871, Japan
e-mail: makinobu@protein.osaka-u.ac.jp
N. Maki
PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi,
Saitama 332-0012, Japan

Current Topics in Microbiology and Immunology (2013) 367: 237–252 237
DOI: 10.1007/82_2012_293
Ó Springer-Verlag Berlin Heidelberg 2012
Published Online: 30 November 2012

........... we introduce a unique phenomenon of transdifferenti- ation identified in newt lens regeneration.. jaw.............................. small intestine. 244 4 Discussion ............................ 2........ One major mechanism of epigenetics is the chemical modifications to the nucleosome. After fertilization. Understanding the mechanism of amphibian regeneration will provide crucial information for both basic and applied biology........ Especially...... 2007).... during newt lens transdifferentiation.. In this chapter.. retina... tail..............and linker-histone regulation................ and contributes to the diversity of gene expression and memory of cell lineage.1 A Key Biological Event In newt lens regeneration............ Kimura 3....................... including DNA methylation and histone modification.. limbs......... Maki and H..... core...... The initial step after lentectomy (from day 0 to day 3) involves ....238 N.......... the field of epigenetics research during regeneration has just started...... the zygote differentiates into diverse cells depending on inter- actions between their cell lineage and differentiation signals............ In this review.............. we focus on two mechanisms of epigenetic changes...............2 Oocyte-Type Linker Histone B4... 2009............ the newt can regenerate almost all tissues in its body including lens... 246 References.... the understanding of unique events in regenerative animals will be important............ dorsal iris pigmented epithelial cells (PECs) transdif- ferentiate into lens cells (Fig 1a). It is clear that epigenetics plays a major role in development........... 248 1 Introduction The developmental program is controlled by genetic and epigenetic regulation..... and brain.......... In particular... they exhibit different profiles of gene expression... Newt lens regeneration can be divided into three major steps........ Sadler et al......... 2007) and during zebrafish pancreatic b-cell and liver regeneration (Anderson et al............ Epigenetic regulation provides the diversity of cell differentiation in development... Epigenetics involves heritable alterations of gene expression without changes in DNA sequence....... Although differen- tiated cells have identical DNA sequences. heart...... There are pioneering studies in DNA methylation during Xenopus limb regeneration (Yakushiji et al.. 2 Newt Lens Transdifferentiation Urodele amphibians have a strong regenerative ability..

a Dorsal PECs transdifferentiate into lens cells. suggesting that lens transdiffer- entiation is accomplished by a different mechanism from that seen in embryogenesis. the PEC nucleus swells and its nucleoli become huge. b Structural change in the nucleus during lens transdifferentiation. After day 14. During dedifferentiation. posterior cells of the dorsal vesicle elongate and start to express lens markers. Embryologically. In the last step. PECs change to transparent cells by around day 8. respectively (Coulombre 1965). Original PECs have a small and shrunken nucleus. After day 14. This process. which is of a considerable size and normal morphology by day 20. nucleus. lens cells and PEC are derived from surface and neural ectoderm origin. The vesicle grows and differentiates into lens. arrow head. About 4 days after lentectomy. Illustration of nucleus is reproduced from (Eguchi 1980) with permission molecular and cellular events that precede PECs re-entering the cell cycle. nucleolus. where PECs lose their original tissue characteristics. lens differentiation begins. On days 10–12 depigmented PECs form a vesicle but do not express lens-specific markers. The transdifferentiation of PECs has been demonstrated in clonal culture experi- ments. PECs begin to re-enter the cell cycle and shed their pigments. N. The next step (days 4–12) is the point at which PECs start cell cycle re-entry 4–5 days after lentectomy. is called dedifferentiation. PECs start shedding their pigment granules. PECs continue depigmentation and proliferation and finally change to transparent cells by day 8. which expresses lens-specific markers in culture (Abe and Eguchi 1977). 1 Newt lens regeneration.Epigenetics and Regeneration 239 Fig. . lens differentiation occurs from dorsal iris. Although the ventral PECs show depigmentation and cell cycle re-entry. they never regenerate lens. At this time. A single PEC dissociated from dorsal iris transdifferentiates into a lentoid body.

Ohmura et al. is highly expressed in stem cells. Therefore during transdifferentiation. the expression of NS is activated and NS accumulates in nucleoli of . It is suggested that the nuclear swelling with the enlargement of euchromatin during the dedifferentiation is due to reprogramming of the cellular state from differentiated to a stem cell-like state. progenitor cells.1 Nucleostemin Nucleostemin (NS). the nucleus dynamically changes its structure in cor- relation with the cellular state. Both knocking down and over-expression of NS reduces cell proliferation in cultured cells. 2009). 2005. 2. increase dramatically (Maki et al. 2. 1b). Although the nucleoli in the original PEC are small. 2009. however. The structure of the nucleoli also changes during the dedifferenti- ation (Fig. 2008. The nucleus of the original PEC is small (about 10 lm in a diameter) and shrunken in shape and has highly developed heterochromatin. To understand the cellular state during newt dedifferentiation. 2010b). expression of NS during the early process of lens regeneration has been analyzed. Maki and H. In contrast to dorsal PECs.240 N. and have small nucleoli.2 Structural Changes in the Nucleus The PEC nucleus dynamically changes its structure during lens transdifferentiation (Maki et al. The number of BrdU-positive cells in the ventral iris is comparable with that in the dorsal iris by day 6 (Maki et al. they become huge during the dedifferentiation. In parallel with the nuclear swelling. After lens removal. 2006). a member of the nucleolar GTPase family. the ventral PECs never differentiate to lens in vivo (Grogg et al. Tsai and McKay 2002) and p53-independent pathways (Beekman et al. 1b). Therefore.3 Gene Expression 2. Romanova et al. 2010b). nuclear swelling occurs. 2010b) (Fig. After the onset of lens differentiation. Hayashi et al. Kimura It is note worthy that the transdifferentiation of PEC is one of the best and most suitable systems to study epigenetics in regeneration because its cell lineage is so simple and so obvious. transcriptionally active regions. 2007. During the dedifferentiation of the PEC. the nuclei of the cells become smaller and elongated. which is the transcriptionally inactive region of Chromatin (Maki et al. and finally the nucleus changes its shape to become round with its diameter reaching more than 20 lm by around day 10. The major function of NS is as a regulator of proliferation in both p53-dependent (Dai et al. 2008. Nikpour et al. there is a dorso-ventral selectivity in lens regeneration. 2003. 2006. and most cancer cells (Baddoo et al. euchromatic regions.3. The ventral PECs show depigmentation and cell cycle re-entry. Tsai and McKay 2002). 2007).

Wernig et al. Takahashi et al. which consists of a histone octamer. 2007). the reprogramming factors. suggesting that the increase of NS accumulating cells is not due to proliferation of pre-existing stem cells but due to changing of the cellular state of PECs during dedifferentiation. genomic DNA is highly organized as chromatin. It has been shown that dedifferentiated cells from the retinal PECs express c-Myc (Agata et al. Although Oct4 and Nanog are not expressed. Sox2 and Klf4 are upregulated at a very early step (day 2). 2005. and c-Myc are expressed in a stage-dependent manner during dedifferentiation of PEC. the increase of NS accu- mulating cells occurs prior to S-phase re-entry.3. expression of the stem cell factors during dedifferentiation of PECs have been examined (Maki et al. H2B. 2007. Klf4. have been screened and pluripotent stem cells have been induced from fibroblasts by introducing these four factors (Okita et al. 2a). By electrofusion with ES cells. In addition to NS. 2001). histone H2A. Klf4. 3 Epigenetics in Newt Lens Transdifferentiation 3. The nucleosome is a basic unit of chromatin. To further investigate newt dedifferentiation. During newt lens regeneration.Epigenetics and Regeneration 241 dedifferentiated cells (Maki et al. The expression of c-Myc reaches a peak at a later stage (day 8). and H4. Another significant property of the ES cell is an ability to reprogram somatic cells (Cowan et al. Linker histones are bound to the linker DNA which is found between nucleosomes and are responsible for forming higher-order chromatin structure (Fig. the expression of those stem cell factors suggests that dedifferentiated cells have a stem cell-like state. Sox2. and the hybrid cells contribute to all three primary layers of chimeric embryos. and c-Myc. On the basis of this fact. suggesting the existence of reprogramming factors in ES cells. a linker histone.2 Stem Cell Pluripotency Factors Embryonic stem (ES) cells are in a pluripotent state which allows them to dif- ferentiate into all types of cells in three germ layers (Evans and Kaufman 1981. and approximately 147 base pairs of DNA wrapped around the histone octamer. 2009).1 Core Histone Modifications In the nuclei of eukaryotic cells. Yu et al. 1993). 2004). Martin 1981). Oct4. H3. somatic cell nuclei can be reprogrammed and express ES cell markers such as Oct4. Core-histones. are . 2007. 2007. Tada et al. Sox2. expres- sion of Sox2 at the lens differentiating stage has been reported (Hayashi et al. Takahashi and Yamanaka 2006. Importantly. 2. 2007). The histone octamer consists of an H3–H4 tetramer and two sets of H2A–H2B dimers (Kornberg 1974). Retinal PECs from chicken embryo dedifferentiate and transdifferentiate into lens cells in culture.

The histone tail is subject to several post- translational modifications (Kouzarides 2007. Maki and H.242 N. Ruthenburg et al. c There are four types of linker- histones identified small basic proteins consisting of a flexible N-terminus called a ‘‘histone tail’’ and a fold domain that interacts with DNA. Acetylation (Ac) and methylation (Me) of lysine residues at N-terminus of histone H3 and H4 are shown. Kimura Fig. b Histone tail modifications. 2007) (Fig. The nucleosome is packed with histone H1 to form higher order chromatin structure. 2b). 2 Chromatin structure and histone modifications. a Approximately 147 base pairs of DNA wrap around a histone octamer consisting of an H3-H4 tetramer and two H2A-H2B dimers to form a single nucleosome. .

TriMeH3K4 and AcH4 (K5.Epigenetics and Regeneration 243 Generally. 2006). 1999. Mikkelsen et al. Wang et al. 3). 2003. Contrary to HATs. TriMeH3K9 is associated with constitutive heterochromatin such as cen- tromeric heterochromatin (Peters et al. 2003. Because this modification is enriched in the genes which should be repressed for proper development (Bracken et al. regulates gene expression. histone acetylation promotes transcriptional activation (Grant et al. histone deacetylases (HDACs) remove an acetyl group from lysine and induce transcriptional silencing (Rundlett et al. Schaft et al. 2002. 16) in the dedifferentiating cell are increased in both irises toward to day 8. 2006. Li et al. 1996. However. Histone acetyl- transferases (HATs) transfer an acetyl group to a lysine residue and thus neu- tralizes the positive charge on lysine. 1999. which is also a mark for gene repression. In contrast to these modifications. AcH3K9. and AcH4 are histone modifications for gene activation. 1996). Nishioka et al. To understand whether histone modifications are involved in dedifferentiation of PECs and dorsal selectivity of lens differentiation. The compre- hensive analysis of methylated histones in the genome reveals that methylated histones are associated with both transcriptionally active and inactive regions (Barski et al. MeH3K9 and MeH3K27 are marks associated with a repressive state (Fischle et al. Those facts suggest that each histone modifi- cation for gene activation is independently regulated during dedifferentiation of . By genome-wide mapping. 8. 2003). Bernstein et al. Histone methylation is involved in various biological aspects. 3). Schiltz et al. TriMeH3K4 (tri-methylated histone H3 lysine 4) and TriMeH3K36 are associated with actively transcribed genes (Krogan et al. 2006). which are repressed for proper embryonic development and cell fate decisions (Bracken et al. it has been shown that TriMeH3K27 is associated with more than 1000 silenced genes. Lachner et al. 2003. the level of TriMeH3K27 increases in the ventral iris. Mendenhall and Bernstein 2008. which are marks for gene repression. 2010b). DiMeH3K9 and TriM- eH3K9. The acet- ylation status of a promoter region. 2007. The lysine residue can be mono-. histone acetylation is a positive mark for gene expression and associated with euchromatin (Shahbazian and Grunstein 2007). 2003. di-. or tri-methylated and each methylation state is associated with a dif- ferent effect on gene expression. Rice et al. As a result. Although not much changes in the dorsal iris. 2007.and di- methylation on H3K9 are related to facultative heterochromatin. In contrast to those active marks. Kuo et al. Guenther et al. TriMeH3K27. the up-regulation of TriMeH3K27 in the ventral iris suggests its partici- pation in inhibition of lens formation from the ventral iris. are almost constant in both irises during dedifferentiation. 2004). TriMeH3K27 is associated with facultative heterochromatin. 2001). shows a significant difference between the dorsal and ventral iris during dedifferentiation (Fig. TriMeH3K4. including HOX genes. 2007. 2002. 1997). whose formation is developmentally regulated depending on cellular differentiation (Rice et al. 2008). AcH3K9 is decreased during the dedif- ferentiation in both irises (Fig. Spencer et al. Schotta et al. Wang et al. 12. 2003. changes in global histone modification have been analyzed (Maki et al. thereby reducing the interaction between DNA and core-histones. 2003). Mono. which is accomplished by a balance between HATs and HDACs.

. H1foo).2 Oocyte-Type Linker Histone B4 Linker histones are classified into four types. while keep them inactivated (Bernstein et al. an active mark. 2006. The combination of such histone modifications. 2007. during newt lens regeneration. This might be due to a difference in the mode of regeneration between dedifferentiation versus stem cell differentiation. Bernstein et al. However. somatic-. testis-. mouse (H1oo. The decrease of AcH3K9 is an interesting point. 3 Summary of changes in global histone modifications of PEC during dedifferentiation in lens regeneration PEC. a repressive mark.and TriMeH3K9 do not change at the same time suggesting that a modification state of H3K9 is not repressive. Recently. Thus. Maki and H. 2010). 2006). and erythrocyte-type linker histones. are co-modified with TriMeH3K4. could be a hallmark of the chromatin state during newt dedifferentiation. It has been reported that in intact zebrafish developmental regulatory genes are silenced by the bivalent modifications and the silenced genes are activated by loss of TriMeH3K27 modification in the fin regeneration (Stewart et al. 3. 2007). Zhao et al. i. the bivalent modification is not observed. histone modifications during dediffer- entiation in different regenerative animals could be investigated in the future. The increasing of TriMeK4 and AcH4 could be related to gene activation for cell cycle reentry and reprogramming of cellular fate during dedifferentiation. 2c).e. The comprehensive analysis of histone mod- ifications shows that a vast majority of genes modified with TriMeH3K27.244 N. in ES cells and that the co-modified fraction is enriched in genes that function during development (Azuara et al. Mikkelsen et al. Bivalent histone modification with TriMeH3K27 and TriMeH3K4 is a remarkable feature of the ES cell. according to their cellular specificity and sequence homology (Fig. cow (H1foo). oocyte-. it has been demonstrated that the zebrafish heart is regenerated by dedifferentiation of cardiomyocytes using a Cre/ lox system (Jopling et al. newt . 2006. Pan et al. Oocyte-type linker histones have been identified in human (referred as H1oo or H1foo). The bivalent histone modification is thought to poise genes for later activation. It should be noted that Di. Kimura Fig. 2007. 2009). increasing levels of TriMeK4 and AcH4 and decreasing levels of AcH3K9.

the intensities of B4 and H1 signals in each nucleus were measured.Epigenetics and Regeneration 245 Fig. Note that the staining intensities of each panel are not comparative because images were processed to show nuclear distribution of each protein. Student’s t test . 200 lm. Bar. ovary. In this condition. Lane 1. The expression of each gene was normalized with that of ribosomal protein L27. 20 lm. and the ratio of B4 to histone H1 was calculated. Using a vivo-morpholino technique. Note that germinal vesicle (GV) was stained by B4 antibody and the nucleus of follicle cells was stained by H1 antibody c immunostaining of iris during lens regeneration. the amount of B4 in dorsal iris during lens regeneration decreased by nearly 50 %.0342. After immunostaining. b Immunostaining of ovary using B4 and H1 antibody. Bar. 4 Oocyte-type linker histone B4 is required for newt lens transdifferentiation. Asterisks indicate a significant difference at p \ 0. lane 2. dorsal iris 10 days after lentectomy. a Detection of B4 protein by Western blot analysis using B4 antibody or neutralized antibody with the antigen. d Changes in the ratio of B4 to histone H1 during lens regeneration. e Knocking down of B4 altered gene expression of key genes of lens differentiation. expression levels of structural and regulatory genes in lens differentiation were analyzed by qPCR.

2004). 1958. 2001. Furthermore. whereas somatic-type histone H1 prevents the remodeling (Saeki et al. the regenerated lens is considerably small because of inhibited proliferation and induced apoptosis. 2005).246 N. 2010). McGraw et al. Maki et al. we have shown a dynamic change of core-histone modifications. Mandl et al. the ratio starts to decreases and reaches a basal level by day 18. 1997. the cellular state during lens transdifferentiation can be dissected in detail. After lens removal. 1998). B4 knockdown represses gene expression of pax6 and MafB. The ratio reaches a peak at day 12. 2010a. such a peak is not observed in the ventral iris. 2004. and almost abolishes expression of c-crystalline. Wilmut et al. only the newt expresses B4 in somatic cells during lens regeneration (Fig. Tanaka et al. such changes have modified the previous concepts of dedifferentiation during the process of lens transdifferentiation. Following nuclear transfer. B4 is reactivated and incorporated into the nucleus of dedifferentiating PECs. During reprogramming. in assembled chromatin in vitro. when lens differentiation occurs. Gao et al. In the past. 4) (Maki et al. somatic-type linker histone H1 is rapidly replaced by oocyte-type linker histone (Becker et al. B4 allows the chromatin to be remodeled by ATP- dependent chromatin remodeling factor. Thus. oocyte-type linker histone disappears in parallel with an initiation of somatic-type linker histone H1 expression. On day 15. a lens differentiation marker (Fig. emergence of oocyte-type linker histone B4. Thus. 2010a). Teranishi et al. Kimura (B4). 4). Ohsumi and Katagiri 1991. Unlike other animals analyzed. the somatic nucleus regains pluripotency to differentiate into all the cell types in the animal (Gurdon et al. when the cells are still undifferentiated. frog (B4. Epigenetic reprogramming occurs after somatic cell nuclear transfer (SCNT) into oocyte. Incorporation of oocyte-type linker histone into the nucleus is required for the reactivation of pluripotency genes such as Oct4 and Sox2 in reprogramming after SCNT (Jullien et al. In fact. Maki and H. The oocyte- type linker histones are predominant linker histones during oogenesis and early embryogenesis. If B4 is knocked down. zebrafish (H1M). The ratio of B4–H1 in dorsal iris PEC starts to increase 8 days after lentectomy. Wakayama et al. and expression of stem cell factors during newt lens transdifferentiation. 2003. However. Moreover. it has been thought that the reprogramming of PEC . 1997. 2005. Wibrand and Olsen 2002). 2006. and sea urchin (cs-H1) (Cho and Wolffe 1994. Using those markers. transcriptional factors in lens differentiation. After the onset of zygotic gene expression. expression of B4 in somatic cells is required in newt lens transdifferentiation and it is sug- gested that reprogramming in the newt somatic cell during transdifferentiation and in the oocyte after SCNT share common strategies. oocyte-type linker histone has a func- tional significance in chromatin remodeling and is required for the reprogramming after SCNT. 4 Discussion In this review. H1X).

Note that these genes are activated sequentially through lens transdifferentiation. we propose a .Epigenetics and Regeneration 247 Fig. shows a peak of expression on day 12. Especially. b New model for reprogramming in newt lens transdifferentiation. However. a Expression prolife of B4 and stem cell factors in dorsal iris PECs during lens transdifferentiation. Based on the expression profile of reprogramming-related genes. this model is not based on the gene expression profile in lens transdifferen- tiation. Thus far. occurs only at an early step of lens regeneration (earl step model). however. 5 Reprogramming in newt lens transdifferentiation. oocyte-type linker histone. which is required for the reprogramming after SCNT. it has been thought that the reprogramming in which differentiated PEC change to stem cell-like cell. we propose a new model in which the somatic nucleus is reprogrammed in a stepwise fashion through the lens transdifferentiation process (whole step model) is completed by about 8 days after lentectomy and that the reprogrammed cells already have a restored ability for lens differentiation (Fig. In fact. Based on the expression profile of those genes. those gene markers related to nuclear reprogramming are expressed sequentially throughout lens transdifferentiation and not just limited to the period prior to cell cycle re-entry. since the cells have lost the morphological characteristics of PEC and have re-entered the cell cycle. 5a).

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