Mechanisms of Development 111 (2002) 153–157
www.elsevier.com/locate/modo
Gene expression pattern
Ilf2 is regulated during meiosis and associated to
transcriptionally active chromatin
L.A. López-Fernández, M. Párraga, J. del Mazo*
Department of Cell and Developmental Biology, Centro de Investigaciones Biológicas, Velázquez, 144, 28006 Madrid, Spain
Received 19 July 2001; received in revised form 31 August 2001; accepted 18 October 2001
Abstract
Analysis of gene expression during testis development demonstrated accumulation of Ilf2 mRNA in pachytene spermatocytes. In these
cells, the protein was localized in the nucleus, but it was absent from chromatin of the XY pachytene bivalent, in which there is no
transcriptional activity. Nucleolar signal is inmmunolocalized in spermatogonia, Sertoli cells and oocytes. By in situ hybridisation, Ilf2
expression is detected in proliferative cells of adult ovary and a defined pattern is also exhibited in different tissues of embryos. The presence
of ILF2 in active chromatin is corroborated in NIH3T3 cultured cells after transfection with Ilf2-EGFP constructs. q 2002 Elsevier Science
Ireland Ltd. All rights reserved.
Keywords: Ilf2; Ilf3; Active chromatin; Meiosis; Spermatogenesis; Testis; Ovary; Pachytene spermatocytes; Oocytes; Mouse embryo
1. Results
1.1. The sequence of Ilf2 is evolutionary conserved and
related to Ilf3
described (Buaas et al., 1999). This suggests that Ilf2 and
Ilf3 are evolutionarily, and probably functionally, related
genes.
1.2. Ilf2 mRNA in testis development
Mouse Ilf2 cDNA was previously isolated from mouse
testis from a subtractive cDNA library (López-Fernández
and del Mazo, 1996). Its 1599 base pairs, (bp.) sequence
(EMBL AC: AJ132273). showed a longest open reading
frame (from 79 to 1249) coding for a protein of 390
amino acids. In vitro translation of Ilf2 cDNA produces a
45 kDa protein as predicted (data not shown). Mouse and
human Ilf2 (AC: AAH00382) show 99.7% of identity in at
aminoacid level, changing only the aminoacid 316. Significant similarities has also been found in Drosophila (AC:
AAF54812) and Caenorhabditis elegans (AC: T24199)
demonstrating a high conservation during evolution (Fig. 1).
ILF2 form a complex with ILF3, which is able to bind the
Antigen Receptor Response Element motif of the IL-2
enhancer (Kao et al., 1994). This ILF2/ILF3 complex has
been described bound to different proteins, such as the
DNA-dependent protein kinase (DNA-PK), acting as a transcriptional regulator (Ting et al., 1998). The dsRNA-dependent protein kinase, PKR has also been described associated
to ILF complex (Langland et al., 1999). Sequence similarity
between human ILF2 and mouse Ilf3 has already been
* Corresponding author. Tel.: 134-91-5644562; fax.: 134-91562-7518.
E-mail address: [email protected] (J. del Mazo).
Northern blots of different adult tissues showed Ilf2
mRNA to be present in ovary, kidney and brain, but is preferentially transcribed in testis (Fig. 2A). Analysis of Ilf2
mRNA in testis at different stages of development revealed
it to be differentially expressed during spermatogenesis (Fig.
2B). Transcripts were first detected at day 15 postnatal (p.n.),
coincident with first developmental differentiation of late
pachytene spermatocytes (Bellvé et al., 1977). Maximum
expression was maintained from day 18 p.n. (when early
round spermatids are present) until adult stage.
To study the putative co-expression of Ilf2 and Ilf3 during
testis development, the same Northern blots were hybridized
with Ilf2 and Ilf3 cDNA probes. The results show an identical
expression pattern for both genes (Fig. 2B). This has also
been observed in a range of adult tissues in which the ubiquitous expression of Ilf3 has been reported, but with the strongest expression in adult testis (Buaas et al., 1999).
In situ hybridisation in testis sections with Ilf2 antisense
riboprobes confirmed the results obtained by Northern blot.
The Ilf2 signal was mainly detected in the basal and proximal layers of the seminiferous tubules (Fig. 3).
To discern which cells showed the most conspicuous
signal of Ilf2, in situ hybridisation was also performed in
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L.A. López-Fernández et al. / Mechanisms of Development 111 (2002) 153–157
positive and negative cells observed for Ilf2and Ilf3. This
pattern could represent the both types of co-existing cells in
corpus luteum: granulosa and lutein. The pattern is more
discernible by a computer-processed image enhancing the
contrast (Fig. 4E).
1.4. Embryo expression
In situ hybridisation with sections from 13 post-coitum
day embryos (E13) showed a defined expression pattern of
Ilf2 and Ilf3 mRNAs (Fig. 4F,G). Signals for Ilf2 and Ilf3
mRNAs were detected in the midbrain, the neopallial cortex
and medial wall of the cerebral hemispheres, the primordium of follicle of vibrissa, the lung, the ovary, trigeminal
ganglion, the kidney, and the medulla.
Fig. 1. Graphical comparison of mouse Ilf2, human ILF2 (AC:
AAH00382), putative Drosophila Ilf2 (AC: AAF54812) and C. elegans
hypothetical protein (AC: T24199). Identity of mouse Ilf2 is higher than
99% with human, 55.6% with Drosophila and 36% with the C. elegans
proteins. Identity is represented by yellow background.
prepuberal testis sections from 20 p.n. day mice. At this
developmental stage, meiosis is complete and round spermatids are present. The different cell types are, in fact, more
discernible than in adult animals. The sections showed Ilf2
to be strongly accumulated in pachytene cells and early
round spermatids (Fig. 3). A weak signal is also observed
in earlier stages.
A similar pattern was found in comparative in situ hybridisation using riboprobes for Ilf3 (Fig. 3).
1.3. Ilf2 and Ilf3 mRNA in ovary
To comparatively analyze expression in female germ
cells, in situ hybridisation was performed in adult ovary
sections. Ilf2 and Ilf3 mRNA accumulation was greatest in
the follicles and corpora lutea (Fig. 4A,B). No signal was
detected in the theca cells of the follicles, but only in growing follicular cells (which later mature into granulosa cells).
In corpus luteum, signal was much more discrete, with both
Fig. 2. (A) Northern blot with RNA from adult tissues (Testis, Ovary,
Kidney, Brain). Blot was hybridised with a mouse Ilf2 probe. (B) Northern
blot with RNA from testes at different times of postnatal development (7,
15, 18, 20, 22, 28 days and adult). Blot was first hybridised with a mouse
Ilf2 probe, and then stripped and hybridised with an Ilf3 probe. Signals were
normalized hybridising the stripped filters with a b-actin probe.
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2. Experimental procedures
2.1. DNA sequencing
Ilf2 cDNA was fully sequenced using both manual and
automatic sequencing methods (Sanger et al., 1977).
Sequences were compared with those in databases using
the Blast and Psi-Blast computer programs (Altschul et
al., 1997). Alignments were performed with the Multalin
program (Corpet, 1988).
2.2. RNA isolation and Northern analysis
Total RNA was extracted from different mouse (Swiss)
tissues by the acid guanidinium method (Chomczynski and
Sacchi, 1987). Northern blots were prepared as previously
described (López-Fernández and del Mazo, 1997) with 20
mg sample loads. Mouse Ilf3 cDNA was obtained from
Genome Systems Inc. (Clone ID: 552078).
2.3. In situ hybridisation
Non-radioactive in situ hybridisation was performed with
tissue cryosections at 12–14 mm thickness mounted on polyl-lysine coated glass slides. Section treatment and hybridisation were performed as previously described (Strahle et
al., 1994). Finally, slides were counterstained with Hoechst
33258, examined using fluorescence and bright field microscopy and photographed.
Fig. 3. In situ hybridisation in testis sections from adult (Ad) and 20 p.n.
days (20d) animals. (A) Digoxigenin-labeled bright field images; (B)
Hoechst-stained sections seen under fluorescence microscopy.
1.5. Nuclear localization Ilf2
Nuclear factors ILF2 and ILF3 have been detected in the
nucleus of Jurkat and 16HBE cells (Aoki et al., 1998; Kao et
al., 1994). Stable transfectants of the mouse cell line
NIH3T3 constitutively expressing the fusion protein Ilf2EGFP, showed Ilf2 to be preferentially localized in nucleoli
(Fig. 5A). In adult testis, Ilf2 was mostly localized in the
nuclei of meiotic cells and less-so in round spermatids (Fig.
5B). However, a strong accumulation of Ilf2 was also
detected in the nucleoli of spermatogonia and Sertoli
cells. Interestingly, the signal in pachytene cells was
completely absent from the XY body (Fig. 5 B4), a typical
conformation of the XY bivalent at pachytene stage where
the chromatin is transcriptionally inactive and without splicing components (Richler et al., 1994).
Ilf2 was also accumulated in the nucleus of oocytes, with
the strongest signal being in the nucleolus (Fig. 5C).
Fig. 4. (A–E) In situ hybridisation in ovary sections from adult mouse of
Ilf2 (A and E, antisense; C, sense) and Ilf3 (B, antisense; D, sense). (E)
Computer-processed image of ovary signals were taken with a CCD
camera. Nuclei are seen in red and digoxigenin signal for Ilf2 mRNA in
green. On star shows a corpus luteum in which there are cells both positive
and negative for digoxigenin label. (F–I) In situ hybridisation of Ilf2 (F,
antisense; H, sense) and Ilf3 (G, antisense; I, sense) in mouse embryo
sections (E13). (NC) Neopallial Cortex, (MB) Midbrain, (TG) Trigeminal
V ganglion, (Lu) Lung, (Ov) Ovary and (K) Kidney.
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2.4. Construction of Ilf2-EGFP fusion protein and
transfections
The 5 0 untranslated region and the open reading frame of
Ilf2 were PCR amplified and subcloned in pGEM-T-easy
(Promega) and digested with SacI and KpnI. A Ilf2-EGFP
fusion protein using pEGFP-N1 vector was generated in
frame.
Cultured mouse NIH3T3 cells were transfected with Ilf2-
pEGFP, pEGFP or without DNA using FuGENE 6 Reagent
(Roche) according to manufacturer’s instructions. Stable
transfectants were selected with Geniticin (Gibco BRL).
Colonies of cells transfected with the Ilf2-EGFP construct
and the positive control EGFP were observed by confocal
microscopy. Images were digitalized and processed using
Adobe Photoshop 4.0 (Adobe Systems).
2.5. Immunohistochemistry
Immunostaining of tissue sections was performed as
previously described (Aoki et al., 1998) with a rabbit antiserum against human ILF2 (1:100), followed by anti-rabbit
biotinated antibody (1:100) and finally developed with
streptavidine-CY3 (1:200) (Amersham). Slides were stained
with Hoescht 33258, mounted and visualized by fluorescence microscopy. Images were recorded with a CCD
camera and processed as above.
Acknowledgements
The authors thank J.F. Escolar for technical assistance
and K. Harshman for English corrections. Antibody aIlf2
was provided by P.N. Kao. This work was supported by
grants from the DGI (BMC2000-1127), EU (PL960183)
and C.A.M. (07B/0022/1997). L.A.L-F was supported by
a C.A.M. fellowship.
References
Fig. 5. (A) Ilf2-EGFP expression in NIH3T3 cells. Ilf2-EGFP fusion protein
is mostly localized in the nucleoli. The white arrows show a mitotic cell
with no signal in condensed chromatin. EGFP signal is observed by excitation with a fluorescent source at 480 nm (1) and cells are observed in a
contrast phase field (2). (B) Immunolocalisation of Ilf2 in adult testis.
Nuclear staining with Hoestch is seen in blue, the aIlf2 antibody signal
in red. Merged of both images of a seminiferous tubule section (1). Different seminiferous tubule sections (2, 3 and 4). White rectangles are augmented below: (2) Sertoli cell with a characteristic prominent nucleolus which
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