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. 2013 Jun 4;6(1):15.
doi: 10.1186/1756-8935-6-15.

Distinct Roles of KAP1, HP1 and G9a/GLP in Silencing of the Two-Cell-Specific Retrotransposon MERVL in Mouse ES Cells

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Free PMC article

Distinct Roles of KAP1, HP1 and G9a/GLP in Silencing of the Two-Cell-Specific Retrotransposon MERVL in Mouse ES Cells

Irina A Maksakova et al. Epigenetics Chromatin. .
Free PMC article

Abstract

Background: In mouse embryonic stem cells (mESCs), transcriptional silencing of numerous class I and II endogenous retroviruses (ERVs), including IAP, ETn and MMERVK10C, is dependent upon the H3K9 methyltransferase (KMTase) SETDB1/ESET and its binding partner KAP1/TRIM28. In contrast, the H3K9 KMTases G9a and GLP and HP1 proteins are dispensable for this process. Intriguingly, MERVL retroelements are actively transcribed exclusively in the two-cell (2C) embryo, but the molecular basis of silencing of these class III ERVs at later developmental stages has not been systematically addressed.

Results: Here, we characterized the roles of these chromatin factors in MERVL silencing in mESCs. While MMERVK10C and IAP ERVs are bound by SETDB1 and KAP1 and are induced following their deletion, MERVL ERVs show relatively low levels of SETDB1 and KAP1 binding and are upregulated exclusively following KAP1 depletion, indicating that KAP1 influences MERVL expression independent of SETDB1. In contrast to class I and class II ERVs, MERVL and MERVL LTR-driven genic transcripts are also upregulated following depletion of G9a or GLP, and G9a binds directly to these ERVs. Consistent with a direct role for H3K9me2 in MERVL repression, these elements are highly enriched for G9a-dependent H3K9me2, and catalytically active G9a is required for silencing of MERVL LTR-driven transcripts. MERVL is also derepressed in HP1α and HP1β KO ESCs. However, like KAP1, HP1α and HP1β are only modestly enriched at MERVL relative to IAP LTRs. Intriguingly, as recently shown for KAP1, RYBP, LSD1 and G9a-deficient mESCs, many genes normally expressed in the 2C embryo are also induced in HP1 KO mESCs, revealing that aberrant expression of a subset of 2C-specific genes is a common feature in each of these KO lines.

Conclusions: Our results indicate that G9a and GLP, which are not required for silencing of class I and II ERVs, are recruited to MERVL elements and play a direct role in silencing of these class III ERVs, dependent upon G9a catalytic activity. In contrast, induction of MERVL expression in KAP1, HP1α and HP1β KO ESCs may occur predominantly as a consequence of indirect effects, in association with activation of a subset of 2C-specific genes.

Figures

Figure 1
Figure 1
MERVL ERVs are derepressed upon KAP1 but not SETDB1 depletion, while MMERVK10C and IAP ERVs are upregulated following depletion of both. (A) Repbase annotations of the LTR and internal regions of full-length MERVL, IAPEz and MMERVK10C elements are shown. Black bars indicate qPCR amplicons for LTRs of each family of element and the internal pol gene region for MERVL. (B) Deregulation of transposable element families in KAP1 and SETDB1 KO mESCs. RNA-seq data for SETDB1 [23] and KAP1 [28] KO lines and the corresponding wt cell lines were used to calculate Z-score values for all annotated retroelements and plotted as shown. (C) KAP1 and SETDB1 were depleted by RNAi in wt TT2 mESCs, and reactivation of MERVL and MMERVK10C elements was determined by qRT-PCR. Mean expression (+/-SD) of each ERV (normalized to β-actin) relative to a scrambled siRNA pool (Scram) is shown for three technical replicates. (D) MERVL is among a small group of transposable elements bound by KAP1 but not SETDB1. RPKM (*10) values generated from published ChIP-seq data for KAP1 [46] and SETDB1 [47] were plotted for all retroelements. Numerous class I and class II ERVs, including IAP subfamilies are enriched for both proteins, which are generally strongly correlated. MERVL elements are modestly enriched for KAP1, but show relatively low levels of SETDB1 coverage. ChIP-seq, chromatin immunoprecipitation sequencing; RPKM, reads per kilobase per million mapped reads.
Figure 2
Figure 2
Unique regions flanking IAPEz but not MERVL elements are highly enriched in KAP1. (A) Profiling of KAP1 in the flanking sequence of all full-length IAPEz, MERVL and MMERVK10C ERVs. KAP1 ChIP-seq reads [46] from wt mESCs were aligned to the mouse genome (mm9), and the density profile of unique reads mapping to the 6 kb regions flanking all annotated intact MERVL (656), MMERVK10C (298) and IAPEz (599) elements, was plotted as shown. (B-C) Heat maps of KAP1 enrichment in the genomic regions flanking 599 IAPEz and 656 MERVL elements in wt mESCs. KAP1 ChIP-seq reads [46] were aligned to the mouse genome (mm9), and the density of uniquely aligned reads, mapping to the 6 kb regions flanking all intact ERVs of the specified families, was plotted. Reads extending into the ERV are due to in silico extension of aligned reads by 300 bp. (D) ChIP and qPCR analysis of KAP1 in TT2 wt mESCs at the LTRs of IAPEz, MMERVK10C and MERVL, as well as the MERVL pol internal region. IgG, negative control IP. Data are mean enrichment from three technical replicates as a percentage of the input chromatin and error bars represent SD. IgG, immunoglobulin G; IP, immunoprecipitation; SD, standard deviation.
Figure 3
Figure 3
MERVL ERVs are derepressed in G9a- and GLP-deficient mESCs and MERVL silencing is dependent on G9a catalytic activity. (A) Upregulation of MERVL in G9a and GLP KO mESCs. Expression of MERVL, MMERVK10C and IAPEz ERVs in TT2 wt, G9a and GLP KO mESCs was analyzed by qRT-PCR. Mean (+/-SD) expression levels relative to the wt line for three technical replicates (normalized to β-actin) are shown. (B) Catalytic activity of G9a but not GLP is required for MERVL silencing. G9a or GLP KO mESCs stably expressing wt or catalytic mutant G9a (C1168A) or GLP (C1201A) [50] transgenes, respectively, were assessed for MERVL expression by qRT-PCR, as described above. (C) MERVL but not MMERVK10C or IAPEz ERVs are upregulated upon KD of Glp. Relative expression of ERVs was determined by qRT-PCR, as described above. (D) Efficiency of each KD was determined by qRT-PCR with primers specific for Kap1 and Glp, as described above. SD, standard deviation.
Figure 4
Figure 4
G9a is bound at MERVL and H3K9me2 enrichment at MERVL elements is dependent on G9a. (A-B) ChIP and qPCR of G9a in TT2 wt and G9a KO mESCs at LTR of MERVL and MERVL internal region and LTRs of IAPEz and MMERVK10C. IgG, negative control IP. Data are mean enrichment from three technical replicates as a percentage of the input chromatin and error bars represent SD. (C-E) N-ChIP and qPCR was performed for H3K9me1 (me1), H3K9me2 (me2), H3K9me3 (me3) and IgG as a negative control in TT2 wt and G9a KO mESCs at the LTRs of MERVL, IAPEz and MMERVK10C. Data are mean enrichment (+/-SD) for three technical replicates normalized to input. IgG, immunoglobulin G; IP, immunoprecipitation; SD, standard deviation.
Figure 5
Figure 5
MERVL ERVs are derepressed in HP1α and HP1β KO mESCs but HP1α and HP1β show relatively low enrichment at these ERVs. (A) RNA-seq analysis of retroelement expression in HP1α and HP1β KO mESCs. RNA-seq data for HP1α and HP1β KO mESCs and the HM1 parent line was generated, and Z-scores were calculated for all retroelements and plotted. Note that MERVL elements show relatively high levels of derepression in both KO lines. (B) MERVL elements are upregulated in HP1α and HP1β KO mESCs. Expression of MERVL, IAP and MMERVK10C ERVs was analyzed by qRT-PCR in HM1 wt, HP1α and HP1β KO lines. Mean (+/-SD) expression levels (normalized to β-actin) are shown, relative to the wt line for three technical replicates. (C-F) IAPEz elements show significantly higher levels of HP1α and HP1β enrichment than do MERVL elements. Cross-linked ChIP was performed with antibodies specific for HP1α or HP1β in HM1 and the corresponding KO cell line. IgG was used as a negative control. The level of enrichment for each IP was determined by qPCR, and the mean and standard deviation of three technical replicates are shown for each experiment. IgG, immunoglobulin G; IP, immunoprecipitation; SD, standard deviation.
Figure 6
Figure 6
Two-cell embryo-specific genes are induced in HP1α, HP1β, KAP1 and G9a KO mESCs. A list of two-cell (2C) specific genes was produced from single blastomere expression data [52] by identifying genes expressed at levels 4-fold higher at the 2C stage than the oocyte or 8C stages (Oo < 2C > 8C). (A-B) Venn diagrams illustrating the overlap between this gene list and the list of genes upregulated at least 4-fold in KAP1, G9a [12,28], HP1α or HP1β KO mESCs are shown. The percentage of genes upregulated in the KO that are also 2C-specific is displayed above, while the percentage of 2C-specific genes that are also upregulated in the KO are presented below. (C) The nine genes upregulated in all four KO lines are listed, along with the distance of the nearest MERVL LTR (MT2) to the transcription start site (TSS). (D) Confirmation of the expression pattern of previously identified 2C-specific genes. RPKM values, derived by division of reads per million (RPM) values from RNA-seq data generated from pooled single blastomeres [52] by transcript length, are presented for Zfp352, Zscan4d, Zscan4c, Tdpoz3 and Tdpoz4 genes. (E) Expression levels of these 2C-specific genes was determined in G9a, KAP1, HP1α and HP1β KO mESCs as well as their wt parent lines using our RNA-seq data (HP1α and HP1β) or previously published RNA-seq data (G9a and KAP1 [12,28]), and Z-score values (see Materials and Methods) for each are presented.
Figure 7
Figure 7
Overview of transcriptional silencing mechanisms acting at different ERV families and the effect of chromatin factor depletion on mESC fate. (A) The mechanism of transcriptional silencing of class I and II ERVs is distinct from that acting on the class III ERV MERVL. KAP1 is recruited to numerous class I and II ERVs, including IAP and MMERVK10C elements, via an interaction between the RBCC domain of KAP1 and the KRAB box of KRAB-ZFPs, which presumably bind directly to specific sequences within these ERVs. The SUMOylated bromodomain of KAP1 recruits SETDB1, which deposits the repressive H3K9me3 mark. MERVL elements in contrast, are bound by G9a and marked by H3K9me2 in a G9a-dependent manner. As these ERVs are upregulated in the absence of G9a or GLP, we propose that the G9a/GLP complex directly regulates MERVL expression via deposition of H3K9me2. (B) Depletion of specific chromatin factors, including KAP1 and HP1 proteins, may promote MERVL expression via indirect effects. MERVL transcripts and MERVL-driven genic transcripts are abundant at the 2C embryonic stage, but are rapidly depleted at subsequent stages, including the blastocyst, from which mESCs are derived. While a small fraction of wt mESCs continually enter a transient state associated with expression of multiple 2C-specific genes, the percentage of these cells increases dramatically in mESCs depleted of KAP1, G9a/GLP and LSD1 [11,12]. One or more of the 2C-specific genes commonly induced in these cells as well as in HP1α and HP1β KO mESCs may indirectly promote transcription of MERVL elements and MERVL LTR-driven genes.

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