Epigenetic control of retrotransposon expression in human embryonic stem cells

58 Pages·2010·2.06 MB·English

MCB Accepts, published online ahead of print on 1 November 2010
Mol. Cell. Biol. doi:10.1128/MCB.00561-10
Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
Macia et al., 2010

1
2 Epigenetic control of retrotransposon expression in
3 human embryonic stem cells
4 Angela Macia1, Martin Muñoz-Lopez1, Jose Luis Cortes1, Robert K. Hastings3,
D
o
5 Santiago Morell1, Gema Lucena-Aguilar1, Juan Antonio Marchal2, Richard M. w
n
lo
6 Badge3, and Jose Luis Garcia-Perez*,1 a
d
e
7 d

f
r
o
8 1,Andalusian Stem Cell Bank, Department of Molecular Embryology. Center for m

h
t
9 Biomedical Research, University of Granada, Spain; 2,Department of Human tp
:
/
/
m
10 Anatomy and Embryology, Institute of Biopathology and Regenerative Medicine
c
b
.
11 (IBIMER), University of Granada, Granada, Spain; 3, Department of Genetics, a
s
m
12 University of Leicester, UK. .o
r
g
/
13 o
n

14 *, Corresponding author: Jose Luis Garcia-Perez Ph D., Andalusian Stem Cell A
p
r
15 Bank, Department of Molecular Embryology. Center for Biomedical Research il 1
0
,

16 Avda Conocimiento s/n, Armilla, Granada 18100, Spain. 20
1
9
17 [email protected], Telf: [phone]; Fax: +34-
b
y

18 958894652 gu
e
s
19 Running title: Expressed L1/Alu elements in hESCs. t
20 Word count: 2107 words (Material and Methods), 4696 words (Introduction,
21 Results, and Discussion).

1
Macia et al., 2010

1 ABSTRACT
2 Long Interspersed Element-1s (LINE-1s or L1s) are a family of non-LTR
3 retrotransposons that predominate in the human genome. Active LINE-1
4 elements encode proteins required for their mobilization. L1-encoded proteins
5 also act in trans to mobilize Short Interspersed Elements (SINEs), such as Alu
D
o
6 elements. L1 and Alu insertions have been implicated in many human diseases, w
n
lo
7 and their retrotransposition provides an ongoing source of human genetic a
d
e
8 diversity. L1/Alu elements are expected to ensure their transmission to d

f
r
o
9 subsequent generations by retrotransposing in germ cells or during early m

h
t
10 embryonic development. Here, we determined that several subfamilies of Alu tp
:
/
/
m
11 elements are expressed in undifferentiated Human Embryonic Stem Cells
c
b
.
12 (hESCs), and that most expressed Alus are active elements. We also exploited a
s
m
13 expression from the L1 antisense promoter to map expressed elements in .o
r
g
/
14 hESCs. Remarkably, we found that expressed Alu elements are enriched in the o
n

15 youngest subfamily Y, and that expressed L1s are mostly located within genes, A
p
r
16 suggesting an epigenetic control of retrotransposon expression in hESCs. il 1
0
,

17 Together, these data suggest that distinct subsets of active L1/Alu elements are 20
1
9
18 expressed in hESCs, and that the degree of somatic mosaicism attributable to L1
b
y

19 insertions during early development may be higher than previously anticipated. gu
e
s
20 t

2
Macia et al., 2010

1 INTRODUCTION
2 The human genome is highly complex in structure, but only ∼1.5% of
3 human DNA has protein coding potential (53). More than 40% of the genome is
4 composed of sequences derived from mobile genetic elements (transposons and
5 retrotransposons) (53). At present, only LINE-1 elements and some SINEs are
D
o
6 actively transposing in the human genome (62). LINE-1 elements (hereafter w
n
lo
7 LINE-1s) are autonomous retrotransposons that constitute ∼17% of human DNA a
d
e
d
8 (53), and recent estimates indicate that an average human genome contains
f
r
o
m
9 around 80-100 sequences that are able to transpose i.e. are retrotransposition-

h
t
t
10 competent LINE1s (hereafter RC-L1s) (19, 71). However these elements vary p
:
/
/
m
11 dramatically in their retrotransposition activity in cell culture based c
b
.
a
12 retrotransposition assays (19). In addition allelic heterogeneity in s
m
.
13 retrotransposition activity (56, 73) and the presence of RC-L1 elements that show o
r
g
/
14 presence / absence polymorphism between individuals (8, 15, 84) implies that o
n

A
15 there can be significant variation in RC-L1 activity between individual genomes. p
r
il
16 An RC-L1 is ~6 Kilobases (Kb) in length (29, 72), and contains a ∼900bp 10
,

2
17 long 5′ untranslated region (UTR) with internal promoter activity (78), two Open 0
1
9

18 Reading Frames (ORFs), a ∼150bp long 3´UTR, and a poly-A tail (72). ORF1 b
y

g
19 encodes a 40 Kilo-Dalton (KDa) protein with RNA binding and nucleic acid u
e
s
t
20 chaperone activity (38, 40, 52, 59, 60). ORF2 encodes a 150-KDa protein with
21 reverse transcriptase (RT) and endonuclease (EN) activities (33, 61). Both
22 proteins are required for the mobilization of L1 within the human genome (65). L1
23 retrotransposition involves the reverse transcription of an mRNA intermediate by

3
Macia et al., 2010

1 a mechanism termed Target Primed Reverse Transcription (TPRT) (25, 26, 55,
2 64). The mobilization of SINEs occurs by a similar mechanism (46), through the
3 use of the LINE-1 encoded ORF2p (28).
4 Alu elements are the most successful human SINEs and they are present
5 at greater than one million copies in the human genome (53). Alu elements are
D
o
6 non-autonomous non-LTR retrotransposons derived from the human gene 7SL w
n
lo
7 (reviewed in (9, 23)), and the average human genome contains ∼6000 active a
d
e
d
8 core Alu elements (12). An Alu core is defined as the ∼280bp region that includes
f
r
o
m
9 both Alu monomers that are capable of retrotransposing in cultured cells, but

h
t
t
10 excludes any flanking genomic 5′ or 3′ regions. p
:
/
/
m
11 Despite the high prevalence of transposable elements in the human c
b
.
a
12 genome and the presence of several LINE and SINE subfamilies in this genome, s
m
.
13 apparently at present only certain members of each class are active o
r
g
/
14 (denominated “young”, “human specific” or “hot” elements, reviewed in (62)). As o
n

A
15 a consequence, L1 and Alu elements can act as insertional mutagens, and p
r
il
16 indeed, many cases of human disease have been caused by such insertions (11, 10
,

2
17 37). In addition to their potential as insertional mutagens, there are many ways in 0
1
9
18 which de novo L1/Alu insertions and L1/L1 or Alu/Alu recombination can impact b
y

g
19 the human genome (reviewed in (11, 23, 37, 44, 50)). Overall, it is estimated that u
e
s
20 between 1/35 and 1/45 newborns harbor a de novo L1 or Alu retrotransposition t
21 event (24, 31, 42, 49). These new events must occur either in parental germ cells
22 or early in embryonic development, prior to the partitioning of the germ cell
23 lineage. Indeed, through the characterization of human mutagenic insertions and

4
Macia et al., 2010

1 the use of mouse models of L1 retrotransposition, it has been revealed that L1
2 retrotransposition can occur in germ cells, during early embryonic development,
3 and in particular somatic tissues (3, 7, 18, 27, 35, 47, 66-68, 80). On the other
4 hand, recent studies have revealed that L1 mobilization processes are a source
5 of genomic variation among humans, with particular impact on our somatic
D
o
6 genome, as revealed by the identification of several de novo L1 insertions in a w
n
lo
7 cohort of lung tumors (10, 27, 31, 42, 45). a
d
e
8 Human embryonic stem cells (hESCs) offer an excellent model to study d

f
r
o
9 biological processes during early human development, as they mimic pluripotent m

h
t
10 cells isolated from the Inner Cell Mass (ICM) of human embryos (79). Several tp
:
/
/
m
11 hESC and human Embryonic Carcinoma (hECs) cell lines express L1
c
b
.
12 retrotransposition intermediates (RiboNucleoProtein particles or RNPs (39, 52, a
s
m
13 58)), and a diverse constellation of L1 mRNAs (representing active and inactive .o
r
g
/
14 subfamilies) are expressed in these cells (35, 41, 58, 74). Furthermore, several o
n

15 cultured hESC lines can support the retrotransposition of engineered LINE-1 A
p
r
16 elements using a cultured cell-based assay (35, 65). There is a growing, but il 1
0
,

17 disparate, set of observations relating to host factors that influence the 20
1
9
18 retrotransposition of Alu and L1, including the differential effect of APOBEC
b
y

19 proteins on the mobility of L1 and Alu (recently reviewed in (21)), the control of L1 gu
e
s
20 expression by DNA methylation in germ cells by DNMT3L, PIWI proteins and t
21 piRNAs (17, 57) as well as single stranded retrotransposon DNA degradation by
22 the exonuclease Trex1 (77). These observations suggest that there is a diverse
23 array of host defense systems that can interfere with L1 retrotransposition.

5
Macia et al., 2010

1 Perhaps most enigmatic of these systems relates to the fact that full-length L1Hs
2 elements contain an active antisense promoter in their 5′UTR (76). Recently it
3 was reported that, in conjunction with the sense L1 promoter, transcripts initiated
4 from the antisense promoter could trigger an RNAi response that attenuates the
5 mobility of L1 in cultured cells (75, 85). Intriguingly, deletion of the L1 antisense
D
o
6 promoter enhances retrotransposition in cultured cells (85), but it has been w
n
lo
7 retained in the vast majority of endogenous active elements, suggesting that it a
d
e
8 has some essential, perhaps regulatory, function. d

f
r
o
9 To characterize a sample of the active retrotransposon “transcriptome” of m

h
t
10 hESCs, we cloned and sequenced expressed Alu elements, and tested their tp
:
/
/
m
11 retrotransposition potential in cultured human cells. We also utilised L1 antisense
c
b
.
12 transcripts (AS-L1), expressed in hESCs, to identify and map expressed L1 a
s
m
13 elements and their host genes. In addition, we found that the antisense promoter .o
r
g
/
14 of L1 is robust over evolutionary time, and that most expressed L1s are located o
n

15 within genes, suggesting epigenetic control of their expression. A
p
r
16 il 1
0
,

2
0
1
9

b
y

g
u
e
s
t

6
Macia et al., 2010

1 MATERIAL AND METHODS
2 Cell culture
3 All reagents were purchased from GIBCO-Invitrogen unless otherwise
4 indicated. The cell lines PA-1 (86) and HeLa-HA were grown as previously
5 described (6). Briefly, cells were passage by standard trypsinization (using a
D
o
6 0.05% stock) and the culture media was: MEM supplemented with 10% Heat w
n
lo
7 Inactivated Fetal Bovine Serum, 1x Non-Essential Aminoacids, and 1mM L- a
d
e
8 Glutamine. 2102Ep (4) and N-Tera2D1 cl1 (N-Tera2D1) (5) cells were grown in d

f
r
o
9 DMEM-high glucose media supplemented with 10% Fetal Bovine Serum, and m

h
t
10 1mM L-Glutamine. Human Embryonic Stem Cell (hESCs) were grown as tp
:
/
/
m
11 previously described (35). hESCs lines H7, H9, and H13B (WA07, WA09, and
c
b
.
12 WA13) were obtained from Wicell and maintained on gelatin-coated plates using a
s
m
13 irradiated Mouse Embryonic Fibroblasts (MEFs) from CF-1 mice, (Chemicon). γ- .o
r
g
/
14 irradiation with a 2100 Cesium source indicator was used to mitotically inactivate o
n

A
15 MEFs. MEFs were used at a density of 25000 cells/cm2. The culture medium for p
r
il
16 hESCs is: DMEM-Knockout supplemented with 4 ng/ml b-FGF, 20% KO serum 1
0
,

2
17 replacement, 1mM L-Glutamine, 50µM β-mercaptoethanol, and 0.1mM Non- 0
1
9
18 Essential Amino Acids. hESCs were manually passage twice a week. b
y

g
19 Transfected hESCs were grown in Matrigel-coated plates (B&D) using MEF u
e
s
20 conditioned media for 24 hours (35). All the cell lines were grown in a humidified t
21 incubator at 37ºC with 7% CO
2.
22 Approval from the Spanish National Embryo Ethical Committee was obtained to
23 work with hESCs.

7
Macia et al., 2010

1
2 Plasmid DNAs
3 Plasmid DNAs were purified using a Midiprep kit from Qiagen, checked for
4 superhelicity by electrophoresis on 0.7% agarose-ethidium bromide gels (only
5 highly supercoiled preparations of DNA (>90%) were used for transfection), and
D
o
6 filtered through a 0.22µM filter. The following plasmids were used: w
n
lo
7 -pRL-SV40, is a 4.8-kb plasmid that contains the coding region of Renilla a
d
e
8 luciferase under the transcriptional control of the early SV40 promoter. It is d
f
r
o
9 cloned in a modified pBSKS II (Stratagene) plasmid that contains a SV40 late m

h
t
10 polyadenylation signal. tp
:
/
/
m
11 -5S-FF, is a 5.7-kb plasmid that contains the 5´UTR region of a human c
b
.
a
12 L1Hs element (L1.3, (71)) cloned in the sense orientation in plasmid pGL3-basic s
m
.
o
13 (Promega). r
g
/

o
14 -5AS-FF, is a 5.7-kb plasmid that contains the 5´UTR region of a human n

A
p
15 L1Hs element (L1.3, (71)) cloned in the antisense orientation in plasmid pGL3- ril
1
0
16 basic (Promega). ,

2
0
1
17 Derivatives of plasmids 5S-FF and 5AS-FF but containing the 5′UTR 9

b
y
18 region from older LINE-1s (L1PA2, L1PA3, L1PA4, L1PA6, L1PA7, L1PA8 and g
u
e
19 L1PA10) were constructed using the same procedure. s
t
20 -pCEP-5´UTRORF2NoNeo, has been described previously(2). It contains
21 a 5.0-kb NotI-BamHI fragment containing the L1.3 5′UTR and L1.3 ORF2 cloned
22 in pCEP4 (Invitrogen).

8
Macia et al., 2010

1 -pAluNF1-neoIII contains a 2.1-kb fragment containing a 7SL promoter, a
2 copy of the NF1 Alu (a Ya5 member) (82), a neoIII self-splicing indicator cassette
3 (30), a thirty-three bp poly(A) tail, and a BC1 transcription termination sequence
4 cloned in pBSKS-II (Invitrogen). An Age I and a BstZ17I sites were introduced in
5 the 5´and 3´ends of the Alu NF1 respectively, to help the cloning of Alu elements
D
o
6 expressed in hESCs. w
n
lo
a
7 -pCEP-heGFP contains the 0.9-kb coding sequence of the humanized d
e
d
8 enhanced green fluorescent protein (hEGFP), which was derived from the f
r
o
m
9 plasmid phrGFP-C (Stratagene) cloned in pCEP4 (Invitrogen).
h
t
t
p
10 Transfection of cultured cells and assays :/
/
m
c
11 HeLa-HA, 2102Ep, N-Tera2D1, and PA-1 cells were transfected using b.
a
s
12 Fugene6 (Roche) as previously described (6, 83). m
.
o
r
g
13 hESCs were transfected by Nucleofection (Amaxa) exactly as described /
o
n

14 (35) using 4x106 cells and 4µg of purified DNA (2µg of pRL-SV40 and 2µg of A
p
r
15 either 5S-FF or 5AS-FF). Luciferase signal was read using the Dual system kit il 1
0
,

16 from Promega. 2
0
1
9
17 The Alu trans-retrotransposition assay in HeLa-HA was conducted in 6- b
y

g
18 well tissue culture plates as previously described (34). Briefly, 4x104/well HeLa- u
e
s
19 HA cells were plated in 6-well tissue culture plates. We used a full plate per Alu t
20 construct to be analyzed. Approximately 14-18 hours after plating, 3 wells from
21 the plate were co-transfected with 0.66µg of a ‘reporter plasmid’ (pAluNF1-neoIII)
22 and 0.33µg of a ‘driver’ L1 that lacks an indicator cassette (pCEP-

9
Macia et al., 2010

1 5´UTRORF2NoNeo). We used 3 µl of Fugene 6 transfection reagent (Roche
2 Biochemical). The remaining 3 wells were co-transfected with equal amounts of
3 an EGFP-reporter plasmid (human renilla green fluorescent protein (pCEP-
4 EGFP), a ‘reporter plasmid’, and a ‘driver’ L1. Seventy-two hours post-
5 transfection, this set of wells was trypsinized and subjected to flow cytometry.
D
o
6 The percentage of green fluorescent (EGFP) cells was used to determine the w
n
lo
7 transfection efficiency of each sample. Seventy-two hours post-transfection, cells a
d
e
8 in the remaining wells were subjected to G-418 selection (400 µg/ml) for 12 days. d

f
r
o
9 The retrotransposition efficiency is expressed as the number of G-418-resistant m

h
t
10 foci/the number of transfected (EGFP-positive) cells. tp
:
/
/
m
11 RNA Extraction and cDNA synthesis c
b
.
a
s
12 Cells were washed twice with 1x-PBS (Invitrogen) and total RNA extracted m
.
o
13 using the TRIzol reagent (Invitrogen). To generate cDNAs, 4µg of total RNA was rg
/

o
14 treated with 100U of RNase-free DNase I (Promega) for 1 hour at 37°C. To n

A
p
15 prevent contamination with genomic DNA, the DNase treatment was repeated ril
1
0
16 twice. Then, 1 µg of RNA was reverse transcribed with MMLV RT (25U, ,

2
0
17 Promega) primed with a 3´-RACE primer for 1 hour at 42°C following 1
9

b
18 manufacturer instructions. The sequence of the RACE primer is as follows: 5´ y

g
u
19 GCGAGCACAGAATTAATACGACTCACTATAGGTTTTTTTTTTTTVN. e
s
t
20 Alu library
21 To generate a library of expressed Alus, RACE primed cDNAs were used
22 in a PCR reaction with primers Outer (5´GCGAGCACAGAATTAATACGACT) and
23 Alu_library (5´ GGTGGCTCACGCCTGTAATCCCAG) in triplicates using High

10