Intranuclear localization of transcription factories and immunoglobulin heavy chain gene alleles during human B-cell maturation
Immunoglobulin heavy chain (IGH) gene locus is expressed monoallelically in human B cells. Aim. To study the role of nuclear organization in regulation of the IGH expression during B-cell differentiation. Methods. Immunofluorescence in situ hybridization on 3D-preserved nuclei (3D immuno-FISH). Resu...
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nasplib_isofts_kiev_ua-123456789-1528272025-02-23T17:19:47Z Intranuclear localization of transcription factories and immunoglobulin heavy chain gene alleles during human B-cell maturation Внутрішньоядерна локалізація транскрипційних фабрик і алелей гену важкого ланцюгу імуноглобуліну в процесі дозрівання В-клітин людини Ядерная локализация транскрипционных фабрик и гена тяжелой цепи иммуноглобулина в процессе дифференцировки B-лимфоцитов Pichugin, A.V. Iarovaia, O.V. Sklyar, I.V. Lacombe, G. Razin, S.V. Fest, T. Lipinski, M. Vassetzky, Y.S. Structure and Function of Biopolymers Immunoglobulin heavy chain (IGH) gene locus is expressed monoallelically in human B cells. Aim. To study the role of nuclear organization in regulation of the IGH expression during B-cell differentiation. Methods. Immunofluorescence in situ hybridization on 3D-preserved nuclei (3D immuno-FISH). Results. Active RNA polymerase II (Pol II) molecules and the IGH locus were detected in the periphery of the nucleoli at some stages of B-cell differentiation. Conclusions. We observed significant changes in the pattern of distribution of RNA polymerase II in the nucleus during B-cell differentiation, but no preferential co-localization of the productive IGH allele with the transcription factories in the vicinity of the nucleolus and in the nucleoplasm was observed. Лише один аллель гену важкого ланцюгу імуноглобуліну (IGH) експресується в В -клітинах людини. Мета. Вивчити роль ядерної організації у регуляції експресії IGH при диференціюванні В-клітин. Методи. Імунофлуоресцентна гібридизація in situ (3D immuno-FISH). Результати. Активна форма РНК-полімерази II (Pol II) і ген IGH були виявлені на периферії ядерець на деяких стадіях диференціювання В-клітин. Висновки. Ми спостерігали значні зміни в характері розподілу РНК-полімерази II в ядрі під час диференціювання В-клітин. При цьому, не спостерігалося мажорної локалізації продуктивного алелі IGH з транскрипційними фабриками ні у безпосередній близькості від ядерець, ні в нуклеоплазмі. Лишь один аллель гена тяжелой цепи иммуноглобулина (IGH) экспрессируется в Б-клетках человека. Цель. Изучить роль ядерной организации в регуляции экспрессии IGH в процессе дифференцировки В-клеток. Методы. Иммунофлуоресцентная гибридизация in situ (3D immuno-FISH). Результаты. Активная форма РНК-полимеразы II (Pol II) и ген IGH были обнаружены на периферии ядрышек на некоторых стадиях дифференцировки В-клеток. Выводы. Мы наблюдали значительные изменения в характере распределения РНК-полимеразы II в ядре во время дифференцировки В-клеток. При этом, не наблюдалось премущественной локализации продуктивного аллеля IGH с транскрипционными фабриками ни в непосредственной близости от ядрышек, ни в нуклеоплазме. This work was supported by the grant #14-24-00022 Russian Science Foundation (RSF). 2016 Article Intranuclear localization of transcription factories and immunoglobulin heavy chain gene alleles during human B-cell maturation / A.V. Pichugin, O.V. Iarovaia, I.V. Sklyar, G. Lacombe, S.V. Razin, T. Fest, M. Lipinski, Y.S. Vassetzky // Вiopolymers and Cell. — 2016. — Т. 32, № 3. — С. 179-183. — Бібліогр.: 18 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.00091C https://nasplib.isofts.kiev.ua/handle/123456789/152827 577 en Вiopolymers and Cell application/pdf Інститут молекулярної біології і генетики НАН України |
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Structure and Function of Biopolymers Structure and Function of Biopolymers |
| spellingShingle |
Structure and Function of Biopolymers Structure and Function of Biopolymers Pichugin, A.V. Iarovaia, O.V. Sklyar, I.V. Lacombe, G. Razin, S.V. Fest, T. Lipinski, M. Vassetzky, Y.S. Intranuclear localization of transcription factories and immunoglobulin heavy chain gene alleles during human B-cell maturation Вiopolymers and Cell |
| description |
Immunoglobulin heavy chain (IGH) gene locus is expressed monoallelically in human B cells. Aim. To study the role of nuclear organization in regulation of the IGH expression during B-cell differentiation. Methods. Immunofluorescence in situ hybridization on 3D-preserved nuclei (3D immuno-FISH). Results. Active RNA polymerase II (Pol II) molecules and the IGH locus were detected in the periphery of the nucleoli at some stages of B-cell differentiation. Conclusions. We observed significant changes in the pattern of distribution of RNA polymerase II in the nucleus during B-cell differentiation, but no preferential co-localization of the productive IGH allele with the transcription factories in the vicinity of the nucleolus and in the nucleoplasm was observed. |
| format |
Article |
| author |
Pichugin, A.V. Iarovaia, O.V. Sklyar, I.V. Lacombe, G. Razin, S.V. Fest, T. Lipinski, M. Vassetzky, Y.S. |
| author_facet |
Pichugin, A.V. Iarovaia, O.V. Sklyar, I.V. Lacombe, G. Razin, S.V. Fest, T. Lipinski, M. Vassetzky, Y.S. |
| author_sort |
Pichugin, A.V. |
| title |
Intranuclear localization of transcription factories and immunoglobulin heavy chain gene alleles during human B-cell maturation |
| title_short |
Intranuclear localization of transcription factories and immunoglobulin heavy chain gene alleles during human B-cell maturation |
| title_full |
Intranuclear localization of transcription factories and immunoglobulin heavy chain gene alleles during human B-cell maturation |
| title_fullStr |
Intranuclear localization of transcription factories and immunoglobulin heavy chain gene alleles during human B-cell maturation |
| title_full_unstemmed |
Intranuclear localization of transcription factories and immunoglobulin heavy chain gene alleles during human B-cell maturation |
| title_sort |
intranuclear localization of transcription factories and immunoglobulin heavy chain gene alleles during human b-cell maturation |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| publishDate |
2016 |
| topic_facet |
Structure and Function of Biopolymers |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/152827 |
| citation_txt |
Intranuclear localization of transcription factories and immunoglobulin heavy chain gene alleles during human B-cell maturation / A.V. Pichugin, O.V. Iarovaia, I.V. Sklyar, G. Lacombe, S.V. Razin, T. Fest, M. Lipinski, Y.S. Vassetzky // Вiopolymers and Cell. — 2016. — Т. 32, № 3. — С. 179-183. — Бібліогр.: 18 назв. — англ. |
| series |
Вiopolymers and Cell |
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179
A. Pichugin, O. Iarovaia, I. Sklyar
© 2016 A. Pichugin et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/),
which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited
UDC 577
Intranuclear localization of transcription factories and immunoglobulin
heavy chain gene alleles during human B-cell maturation
A.V. Pichugin1,2,3, O. V. Iarovaia1,2,4, I. V. Sklyar1,2,4, G. Lacombe5,
S. V. Razin2,4,6, T. Fest5, M. Lipinski1,2, Y. S. Vassetzky1,2,6
1 CNRS UMR 8126, Universit Paris-Sud 11, Institut Gustave Roussy
114, rue Edouard Vaillant, Villejuif, France, 94805
2 LIA 1066 French-Russian Joint Cancer Research Laboratory
Villejuif, France–Moscow, Russian Federation
3 Peter the Great St. Petersburg Polytechnic University
29, Polytechnicheskaya Str, St.Petersburg, Russian Federation, 195251
4 Institute of Gene Biology, Russian Academy of Sciences
34/5, Vavilova Str., Moscow, Russian Federation, 119334
5 INSERM U917, Université de Rennes
2, avenue du Professeur Léon Bernard F - 35043 Rennes, France
6 Faculty of Biology, M. V. Lomonosov Moscow State University
Leninskie Gory, Moscow, Russian Federation, 119991
vassetzky@igr.fr
Immunoglobulin heavy chain (IGH) gene locus is expressed monoallelically in human B cells. Aim. To study the
role of nuclear organization in regulation of the IGH expression during B-cell differentiation. Methods. Immuno-
fluorescence in situ hybridization on 3D-preserved nuclei (3D immuno-FISH). Results. Active RNA polymerase
II (Pol II) molecules and the IGH locus were detected in the periphery of the nucleoli at some stages of B-cell
differentiation. Conclusions. We observed significant changes in the pattern of distribution of RNA polymerase
II in the nucleus during B-cell differentiation, but no preferential co-localization of the productive IGH allele with
the transcription factories in the vicinity of the nucleolus and in the nucleoplasm was observed.
K e y w o r d s: Immunoglobulin heavy chain gene, transcription, nucleolus, B-cell maturation
Introdution
Nucleus is divided into transcriptionally active and
repressive compartments (reviewed in [1]).
Transcriptional activation is associated with the eu-
chromatic regions of the nucleus and transcriptional
factories (reviewed in [2]). A functional immuno-
globulin heavy chain is produced monoallelically
(Rajewsky 1996); it undergoes regulated transcrip-
tion during the B lymphocyte development.
Transcription might be controlled by localized al-
terations in the chromatin structure. Whether such a
mechanism is sufficient to account for the allele-spe-
cific transcription of the IGH locus is currently un-
known. In mice, the localization of productive and
non-productive IGH alleles changes during B-cell
maturation. A non-productive IGH allele is located
close to the nuclear periphery whereas the produc-
tive IGH allele is associated with euchromatin [5–7];
much less is known about the role of nuclear organi-
zation in the human IGH allelic transcription. We
have recently found that the IGH locus was located
ISSN 1993-6842 (on-line); ISSN 0233-7657 (print)
Biopolymers and Cell. 2016. Vol. 32. N 3. P 179–183
doi: http://dx.doi.org/10.7124/bc.00091C
180
A. Pichugin, O. Iarovaia, I. Sklyar et al.
close to the nucleolus in both naïve and activated
B-cells [8] as well as in B-cell lymphoma [9].
However, the functional significance of this localiza-
tion remains unknown.
Given the evidence that the eukaryotic gene acti-
vity can be regulated by subnuclear compartmenta-
lization (reviewed in [10]), we investigated whether
the perinucleolar localization of the IGH locus might
play a role in controlling its transcription. Here we
have used 3D-fluorescence in situ hybridization
(3D-FISH) combined with immunofluorescence to
co-detect the localization of the IGH alleles, nucleo-
li and transcription factories at different stages of B
lymphocyte maturation.
Materials and Methods
In vitro B-cell differentiation
Peripheral blood cells from healthy volunteers were
purchased from the French Blood Center. B-cells at
different stages of differentiation were obtained es-
sentially as described in [11]. Plasmocytes were iso-
lated from peripheral blood using the CD138+
Plasma Cell Isolation Kit (Miltenyi Biotech).
Three-dimensional fluorescence in situ hybridi-
zation (3D-FISH) and immuno-detection
3D FISH experiments were essentially carried out as
described elsewhere [9]. The stained probes used in
this study (RP11-346I20 that recognizes the constant
region of the IGH locus and RP11-259B19 for the
J/D region) were purchased from Blue Gnome
(Cambridge, UK). Nucleoli were detected using
mouse anti-B23 antibody (Sigma, St Louis, MO)
and chicken anti-mouse Alexa 647 (Molecular
Probes, Carlsbad, CA). The Pol II antibodies were
from Active Motif, Carlsbad, CA. The images were
processed and analyzed as described in [9].
Results and Discussion
In order to study whether the IGH localization vs.
transcription factories undergoes dynamic changes
linked to its functional status, we have followed the
localization of the IGH alleles in human cells during
B-cell maturation in vitro [11]; this system allows
reproducing the major stages of B-cell differentia-
tion in lymph nodes. We used B-cells at the follow-
ing stages: naive B-cells (Day0), activated B-cells
(Day1), cells at the stage of plasmoblast differentia-
tion (Day4), and cells at the stage of somatic hyper-
mutation and class switch recombination (Day5/6).
The IGH locus being located close to a nucleolus
at some stages of B-cell maturation [8], we wondered
whether its transcription would actually take place in
the perinucleolar region which, with some exceptions
[9] is not known for being prone to [the] RNA poly-
merase II (Pol II)-dependent gene transcription [12].
To test this, B-cells were stained using the B23 anti-
body that decorates nucleoli and the antibody to the
phosphorylated C-terminal domain of the transcrip-
tionally-active form of Pol II. These active Pol II
molecules are present in nuclear foci called transcrip-
tion factories, where active transcription takes place
(for review see [2]). Typical Pol II staining patterns
are presented in Fig. 1. Uniform staining of active Pol
II molecules was detected in the immediate vicinity
of nucleoli in naïve B lymphocytes at Day0. Activation
of B-cells at Day1 led to a decrease in Pol II staining
with transcription factories being organized in clus-
ters, some of them adjacent to the nucleolus. At Day4,
Pol II was excluded from the perinucleolar regions;
at Day5/6, this exclusion zone disappeared and intense
Pol II staining surrounded the nucleolus. Finally, in
plasmocytes, Pol II staining concentrated in the cen-
ter of the nucleus, with some staining around the nu-
cleolus (Fig. 1).
Next, the proximity between the IGH alleles and
Pol II foci at different stages of B-cell differentiation
was evaluated using an immuno-FISH approach
whereby nucleoli, Pol II clusters, and the productive
IGH alleles were all simultaneously visualized. [The]
Signals from the productive IGH allele (green), and
Pol II (white) were scored in relation to their proxim-
ity to the nucleolus (blue). The results are summarized
in Fig. 2. These data indicate that the productive IGH
allele is preferentially colocalized with the transcrip-
tion factories at the nucleolus in naïve B-lymphocytes
at Day0 and at the SHM and CSR stages at Day5/6.
181
Intranuclear localization of transcription factories and IGH gene alleles during human B-cell maturation
B-cells are monospecific, i.e. they produce one anti-
body per cell. This is due to allelic exclusion of im-
munoglobulin (Ig) heavy (H) and light (L) chain genes
established during V(D)J recombination (reviewed in
[13]). While the somatic generation of functional Ig
genes by V(D)J recombination is a subject to allelic
exclusion, the expression of Ig loci per se does not ap-
pear to be monoallelic as Ig transcripts are expressed
from both alleles whereas only one of the two Ig al-
leles is functional [14]. The molecular and cellular
mechanisms regulating allelic exclusion during V(D)J
recombination are well studied [15], much less is
known on how these mechanisms operate at the final
stages of B-cell differentiation. DNA methylation [16],
chromatin organization [17] and subnuclear localiza-
tion [5–8] might contribute to this process.
The positioning of one allele in the vicinity of a
nucleolus, a statistically significant difference be-
Fig. 2. Association of IGH al-
leles with the RNA poly-
merase II during B-cell matu-
ration. A, quantitation of co-
localization of the productive
(red) and non-productive
(blue) IGH alleles with the
nucleolus and transcription
factories. B, Representative
images of the productive
(green) and non-productive
(merged green and red) IGH
alleles with nuclei stained for
nucleolus (bleu) and RNA
polymerase II (gray) at differ-
ent stages of B-cell matura-
tion. Scale bar=5µm and 1µm
in zoom.
Fig. 1. Localization of transcription factories and nucleoli during induced B-cell maturation. Active RNA polymerase II (green) and
nucleoli (B23, blue) were simultaneously revealed by immunostaining. Typical images are shown. Scale bar=5µm
182
A. Pichugin, O. Iarovaia, I. Sklyar et al.
tween the two alleles, may be linked to the IGH ex-
pression. Indeed, transcriptionally active Pol II clus-
ters could be observed in the vicinity of the nucleolus
in lymphoid cells [9]. It is also known that nuclear
factors localized in and around the nucleolus, such as
nucleolin, are essential for [the] IGH transcription
[18]. Using immuno-FISH experiments, we analyzed
the localization of the two alleles with clusters of
transcriptionally active Pol II molecules (transcrip-
tion factories, see [2] for review). We could not ob-
serve any preferential localization of the productive
IGH allele with the transcription factories in the vi-
cinity of the nucleolus and in the nucleoplasm. At the
same time, the pattern of distribution of RNA poly-
merase II changed during B cell maturation, with the
most striking feature being the exclusion of active
RNA Pol II molecules from the perinucleolar region
in activated B cells at Day4. We have also detected
the presence of active Pol II clusters in the periphery
of nucleoli that in normal cells is mostly occupied by
heterochromatin [12] and therefore is not prone to the
Pol II transcription. Further studies are necessary to
establish a link between transcriptional activity and
subnuclear organization of the IGH alleles.
Acknowledgements
This work was supported by the grant #14-24-00022
Russian Science Foundation (RSF).
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Внутрішньоядерна локалізація транскрипційних
фабрик і алелей гену важкого ланцюгу імуноглобуліну
в процесі дозрівання В-клітин людини
А. В. Пичугін, О. В. Ярова, І. В. Скляр, Г. Лякомб,
С. В. Разин, Т. Фест, М. Липинскій, Є. С. Васецький
Лише один аллель гену важкого ланцюгу імуноглобуліну (IGH)
експресується в В-клітинах людини. Мета. Вивчити роль ядер-
ної організації у регуляції експресії IGH при диференціюванні
В-клітин. Методи. Імунофлуоресцентна гібридизація in situ
(3D immuno-FISH). Результати. Активна форма РНК-полі ме-
ра зи II (Pol II) і ген IGH були виявлені на периферії ядерець на
деяких стадіях диференціювання В-клітин. Висновки. Ми
спостерігали значні зміни в характері розподілу РНК-полі ме-
ра зи II в ядрі під час диференціювання В-клітин. При цьому, не
спостерігалося мажорної локалізації продуктивного алелі IGH
з транскрипційними фабриками ні ву безпосередній близькості
від ядерець, ні в нуклеоплазмі.
К л юч ов і с л ов а: гени важких ланцюгів імуноглобуліну,
транскрипція, ядро, дозрівання B-клітин
Внутриядерная локализация транскрипционных
фабрик и аллелей гена тяжелой цепи иммуноглобулина
в процессе созревания В-клеток человека
А. В. Пичугин, О. В. Яровая, И. В. Скляр, Г. Лякомб,
С. В. Разин, Т. Фест, М. Липинский, Е. С. Васецкий
Лишь один аллель гена тяжелой цепи иммуноглобулина (IGH)
экспрессируется в Б-клетках человека. Цель. Изучить роль
ядерной организации в регуляции экспрессии IGH в процессе
дифференцировки В-клеток. Методы. Иммунофлуоресцентная
гибридизация in situ (3D immuno-FISH). Результаты. Активная
форма РНК-полимеразы II (Pol II) и ген IGH были обнаружены
на периферии ядрышек на некоторых стадиях дифференци-
ровки В-клеток. Выводы. Мы наблюдали значительные изме-
нения в характере распределения РНК-полимеразы II в ядре во
время дифференцировки В-клеток. При этом, не наблюдалось
премущественной локализации продуктивного аллеля IGH с
транскрипционными фабриками ни в непосредственной бли-
зости от ядрышек, ни в нуклеоплазме.
К л юч е в ы е с л ов а: гены тяжелых цепей иммуноглобули-
на, транскрипция, ядро, дозревание B-клеток
Received 14.02.2016
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