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. Author manuscript; available in PMC: 2015 Jan 16.
Published in final edited form as: Cell Rep. 2014 Jan 2;6(1):222–230. doi: 10.1016/j.celrep.2013.12.015

Epigenetic Regulation of the Apolipoprotein Gene Cluster by a Long Non-Protein-Coding RNA

Paul Halley 1,+, Beena M Kadakkuzha 1,+, Mohammad Ali Faghihi 1, Marco Magistri 1, Zane Zeier 1, Olga Khorkova 2, Carlos Coito 2, Jane Hsiao 2, Matthew Lawrence 3, Claes Wahlestedt 1,*
PMCID: PMC3924898  NIHMSID: NIHMS550546  PMID: 24388749

Abstract

Apolipoprotein A-1 (APOA1) is the major protein component of high-density lipoprotein (HDL) in plasma. We have identified an endogenously expressed long non-coding natural antisense transcript, APOA1-AS, which acts as a negative transcriptional regulator of APOA1, both in vitro and in vivo. Inhibition of APOA1-AS in cultured cells resulted in the increased expression of APOA1, and two neighboring genes in the APO cluster. Chromatin immunoprecipitation (ChIP) analyses of a ~50Kb chromatin region flanking the APOA1 gene demonstrated that APOA1-AS can modulate distinct histone methylation patterns that mark active and/or inactive gene expression, through the recruitment of histone-modifying enzymes. Targeting APOA1-AS using short antisense oligonucleotides also enhanced APOA1 expression in both human and monkey liver cells, and induced an increase in hepatic RNA and protein expression in African green monkeys. The results presented here further highlight the significant local modulatory effects of long non-coding antisense RNAs, and demonstrate the therapeutic potential of manipulating the expression of these transcripts both in vitro and in vivo.

Introduction

In recent years, long non-coding RNAs (ncRNAs) have been shown to play important functional roles as regulators of gene expression (Ansari, 2009; Faghihi et al., 2010; Huarte et al., 2010; Katayama et al., 2005; Martinho et al., 2004; Modarresi et al., 2012), through the recruitment of the complex epigenetic machinery that dictates distinctive chromatin signatures involved in active transcription (Kaikkonen et al., 2011; Magistri et al., 2012; Rinn et al., 2007; Tsai et al., 2010a; Wang et al., 2011b). One such group of lncRNAs is the natural antisense transcripts (NATs), which are transcribed from the opposed DNA strand to their specific partner protein-coding (or non-coding) genes. The most common example of antisense transcription is the pairing of a NAT with an overlapping protein-coding (sense) transcript, whereby NAT expression can lead to an increase (concordant) or decrease (discordant) in sense expression (Faghihi and Wahlestedt, 2009). The modulatory effect of an antisense ncRNA on the neighboring genes has been reported in yeast (Camblong et al., 2007) and in mammalian imprinting (Nagano et al., 2008; Sleutels et al., 2002) suggesting that the regulatory role of NATs can also extend beyond sense partner to the overlapping chromatin region.

The observation that more than 70% of mammalian transcriptional units show evidence of antisense transcription, not only indicates the biological importance of NATs, but could also have various therapeutic implications (Katayama et al., 2005; Lehner et al., 2002; Wahlestedt, 2006). For example, identifying and inactivating a discordantly-acting NAT, using various RNA interference approaches, could lead to de-repression and subsequent “switching on” of a gene of interest (Wahlestedt, 2006). Very recently, our group has successfully used this approach to substantially increase the expression of the therapeutic target brain-derived neurotrophic factor (BDNF), through inhibition of endogenous non-coding antisense transcripts that repress BDNF transcription (Modarresi et al., 2012). We have now identified a NAT for another therapeutically relevant gene, the Apolipoprotein A1 (APOA1).

APOA1 is the major protein component of high-density lipoprotein (HDL) in plasma (Barbaras et al., 1987) and is synthesized primarily in the liver (80%) and small intestine (10%) (Elshourbagy et al., 1985). It plays a key role in reverse cholesterol transport, promoting cholesterol efflux from tissues by acting as a cofactor for the lecithin cholesterol acyltransferase (Glomset, 1968). A low HDL cholesterol concentration reflects increased susceptibility to cardiovascular diseases, and raising HDL pharmacologically remains a proposed strategy to reduce the occurrence of cardiovascular diseases (Green et al., 1979;Livshits et al., 1997; Rader, 2002). The genes encoding human APOA1, as well as apolipoproteins C3, A4 and A5, are clustered on chromosome 11q23.3 (Figure 1A), with APOA1, APOA4 and APOA5 transcribed 5′ to 3′ and APOC3 transcribed in the opposite direction (Antonarakis et al., 1988). While previous studies have shown that transcription at the human apolipoprotein gene cluster (A1/C3/A4/A5) is dependent on specific chromatin structures, such as the CTCF/cohesion chromatin insulators (Mishiro et al., 2009), relatively little is known about the epigenetic factors influencing APOA1 expression. Here we report a NAT-mediated mechanism of APOA1 transcriptional regulation that involves the recruitment of multiple chromatin modifying complexes to the APO gene cluster. We demonstrate that targeting this NAT, using both small interfering RNAs (siRNAs) and antisense oligonucleotides (ASOs), can induce an increase in APOA1 expression both in vitro and in vivo respectively.

Figure 1. APO gene cluster and APOA1-AS NAT organization on human chromosome 11.

Figure 1

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The direction of transcription is indicated by arrows.

(A) The antisense transcript APOA1-AS (EST DA327409) has two exons and the first exon is partially overlapping with the forth exon of APOA1 and the second exon is overlapping with the intronic region of QSK gene.

(B) The splice variants obtained from 3′ and 5′ RACE using primers based on DA327409 sequence.

Results

Characterizing an overlapping antisense transcript at the Apo gene cluster

APOA1 mRNA is transcribed from the negative strand of chromosome 11 and contains 4 exons. A potential APOA1-natural antisense (accession number DA327409) EST was identified, using UCSC genome browser (http://genome.ucsc.edu) (Figure S1), that is transcribed from the positive strand of the APOA1 locus and has two exons, positioned ~20 kb apart (Figure 1A). This APOA1 antisense transcript (APOA1-AS) shares a 123 nucleotide-long region overlapping with the fourth exon of APOA1 mRNA (Figure 1A). In order to find the complete sequence, e.g. transcription start site (TSS), alternative splicing and 3′ end of APOA1-AS transcript, 5′ and 3′ RACE experiments were performed using primers designed based on EST sequence. 5′ and 3′ RACE expanded the EST sequence from both 3′ and 5′ ends and determined splice variants with additional 3′exons (Figure 1B). We next examined a panel of RNAs from human tissues for the presence of APOA1 and APOA1-AS transcripts by quantitative RT-PCR, using specifically designed probes. Both APOA1 and APOA1-AS transcripts were expressed in all tissues examined; however, there were significant differences in their expression levels (Figure 2A). APOA1 mRNA was highest in the liver, an order of magnitude less in small intestine and two orders of magnitude less in colon. APOA1-AS was highly expressed in ovary, cervix, testis and thyroid. The ratio of APOA1/APOA1-AS was also seen to vary for many of the tissues examined (Figure 2B). Liver, small intestine and colon showed 103-fold higher expression levels of APOA1 mRNA compared to APOA1-AS, while testis, heart and 12-week embryo showed a 102-fold difference. The APOA1/APOA1-AS ratios were 1 or less than 10-fold in thymus, ovary, spleen, kidney, esophagus, thyroid, adipose tissue, skeletal muscle, placenta, lung, prostate, trachea and brain.

Figure 2. Characterization of the expression profiles of APOA1 and APOA1-AS.

Figure 2

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(A) Quantitative RT-PCR analysis of APOA1 and APOA1-AS on RNA samples from a panel of different human tissues; each transcript was normalized to value of the same transcript in 12-week embryo. (B) The ratio of APOA1 to APOA1-AS transcripts were measured in commercial RNAs obtained from various tissues. (Graphs represent mean relative expression values ± SEM)

APOA1-AS transcript acts as a ‘temporal switch’ to regulate APOA1 expression

The liver expresses large amounts of APOA1, accounting for more than 70% of circulating APOA1 protein in the blood (Eisenberg, 1984). In order to identify any direct link between the sense-antisense expression levels of APOA1 and APOA1-AS transcripts, we next treated liver HepG2 cells with specific siRNAs designed against APOA1-AS. The sequences of all three siRNAs and the areas of the APOA1-AS transcript targeted by each are presented in Figure S1. While all three siRNAs tested showed significant knockdown of APOA1-AS (Figure S2), siRNA 1 showed the highest efficiency (~65%), leading to ~ 3 fold up-regulation of the APOA1 transcript (Figure 3A) and was thus used in all subsequent studies. A time-course experiment was performed, whereby total RNA was extracted at 5, 12, 24, 48 and 72 hours after transient transfection with the APOA1-AS siRNA. APOA1-AS transcript levels were significantly reduced after 5 hours (up to 90%) (Figure 3B) and returned to ~ 60% compared to negative control siRNA treatment after 24 h. APOA1 mRNA concentration was increased over the time course from 12 hours and reached plateau at 72 hours (Figure 3B). The up-regulation pattern of APOA1 mRNA over 72 hours indicates that transient reduction of APOA1-AS expression is sufficient for initiating the transcriptional up-regulation of APOA1 gene, achieving 3-fold elevation at 48 and 72 hours. These results indicate a novel functional role of endogenous APOA1-AS, whereby it can act analogous to a molecular switch to dictate the expression levels of the APOA1 sense gene.

Figure 3. siRNA-mediated down-regulation of APOA1-AS.

Figure 3

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(A) HepG2 cells were transfected with APOA1-AS siRNA and control siRNA for 48 hours and the levels of APOA1 and APOA1-AS genes were measured with quantitative RT-PCR. See also figures S1, S2 and S3 (B) HepG2 cells were transfected with APOA1-AS siRNA, total RNA was collected after 5, 24, 48 and 72 hours, and the transcript levels of APOA1 and APOA1-AS were measured with quantitative RT-PCR. (C) HepG2 cells were transfected with APOA1-AS siRNA and control siRNA for 48 hours. Quantitative RT-PCR was used to measure the expression of the genes belonging to the APO gene cluster. (Graphs represent mean relative expression values ± SEM)

APOA1-AS modulates multiple genes in the APO gene cluster

We hypothesized that APOA1-AS may modulate transcription regulation not only of APOA1 but also other members of the APO gene cluster (C3/A4/A5) located in close proximity on the chromatin. Quantitative PCR analysis of APOA1/C3/A4/A5 transcripts in HepG2 cells transfected with the APOA1-AS siRNA showed that APOC3 and APOA4 were up-regulated ~3 and ~4 fold respectively, but APOA5 mRNA concentration was unchanged (Figure 3C). To further test the extent of APOA1-AS mediated transcriptional regulation, we measured the expression of the neighboring inosine-guanosine kinase (QSK), a serine kinase located 6 kb upstream of APOA1 gene, which has intronic overlap with the second exon of APOA1-AS (Figure 1A). Interestingly, QSK concentration was not affected by the APOA1-AS knockdown, indicating a degree of locus specificity and a possible chromatin boundary region relating to APOA1-AS activity. To evaluate the trans- acting properties of the APOA1-AS, Apolipoprotein B (APOB), another member of the APO gene family that is transcribed from chromosome 22 and is also involved in cholesterol pathway, was also examined (Figure 3C). We did not observe any change in APOB mRNA concentration after knockdown of APOA1-AS, suggesting this regulation of the APO gene cluster by APOA1-AS occurs locally (i.e. in cis-). Together, these findings highlight the significant role played by a cis- acting NAT in local chromatin regulation.

The APO gene cluster is epigenetically regulated by APOA1-AS

We next hypothesized that APOA1-AS could function as a mediator of suppressive epigenetic markers, through the recruitment of chromatin-modifying complexes. To examine APOA1-AS-associated chromatin modifications at the APO gene cluster, we performed chromatin immunoprecipitation (ChIP) experiments to measure the levels of tri-methylated lysines 4, 9 and 27 on histone H3 following siRNA-mediated APOA1-AS knockdown. H3K4-met3 marks transcriptionally active chromatin, while H3K9-met3 and H3K27-met3 are considered to be repressive chromatin marks (Kouzarides, 2002; Strahl and Allis, 2000). Eight primer sets were designed to span a 50 kb region encompassing APOA1, APOC3, APOA4 and APOA5 (Figure 4A, Table S1).

Figure 4. ChIP analysis for histone modifications on the 50 kb chromatin region of APO gene cluster.

Figure 4

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Upper panel: Schematic representation of the ChIP primer sets covering ~50 kb chromatin region of Apo gene locus. See also Table S1.

Lower panel: HepG2 cells were transfected with APOA1-AS siRNA and control siRNA for 48 hours. Cells were lysed and chromatin collected for ChIP analysis of: (A) H3K4-met3 (B), H3K27-met3 (C) H3K9 met3 (D) LSD1 (E) SUZ12 levels along the ~50 kb chromatin region. (F) Background ChIP observed with a control IgG antibody (n=3; * p<0.05; ** p<0.01; ***p<0.001, 2-tailed t-test)

ChIP, followed by quantitative PCR analysis, showed distinctive changes in H3K4-met3 and H3K27-met3 levels, while not significance changes were observed in H3K9-met3. The amplicons amplified by primer set 1–5 (P1–P5) demonstrated significantly elevated levels of the active marker H3K4-met 3 in HepG2 cells treated with the APOA1-AS siRNA (Figure 4B). These amplicons spanned the promoter of APOA1, as well as a specific enhancer element involved in the regulation of APOA1/C3/A4, thus indicating increased transcriptional activity (Figure 4B). In support of this, levels of the repressive mark H3K27-met3 for the P1–P6 region, which also included the promoters for APOC3 and APOA4, were significantly reduced (Figure 4C), while H3K9-met3, another mark associated with gene silencing was unchanged (Figure 4D). It is interesting to note that P6, which represented the amplicon at the promoter of APOA4, demonstrated no change in H3K4 trimethylation (Figure 4B) but a rather pronounced reduction in the levels of repressive chromatin marker H3K27-met3 (Figure 4C).

It was shown that ncRNAs act as scaffold for the locus-specific recruitment of functionally related chromatin modifying enzymes (Guttman et al., 2011; Tsai et al., 2010b). In order to explain the observed changes in histone modifications, further ChIP experiments were performed using antibodies for (1) the lysine (K)-specific demethylase 1 (LSD1), a nuclear protein that is known to induce gene silencing through the removal of active methyl marks, primarily from H3K4 (Shi et al., 2004), and (2) the Suppressor of Zeste 12 homolog (SUZ12), a key component of the polycomb recessive complex (PRC2), which has been shown to mediate chromatin silencing through H3K27 trimethylation (Cao et al., 2002; Kuzmichev et al., 2002). In addition to the increase in active H3K4me3 at the APOA1 promoter (P1–P5) following siRNA treatment, these amplicons (P1–P4) also exhibited significantly reduced LSD1 occupancy (Figure 4E), indicating that APOA1-AS, at least in part, induces the transcriptional silencing of the APOA1 gene through LSD1 recruitment. Furthermore, the decrease in repressive H3K27me3 marks on the amplicons for P1–P6 following siRNA treatment also coincided with a marked reduction of SUZ12 occupancy for this region, suggesting that the observed increase in APOA4 and APOC3 gene expression is due to a disruption of APOA1-AS-mediated PRC2 interaction. ChIP experiments were also carried out for the same samples using an IgG control antibody, to confirm that all chromatin immunoprecipated was specific to LSD1 or SUZ12 and could not be attributed to background, non-specific binding (Figure 4F).

Antisense oligonucleotides targeting APOA1-AS induce APOA1 up-regulation in vitro and in vivo

Given the clear therapeutic potential for the treatment of coronary artery disease, we further tested our hypothesis by examining whether targeting APOA1-AS could promote hepatic APOA1 expression in vivo. Over 80 phosphorothioate-backbone antisense oligonucleotides were designed to cover the full APOA1-AS sequence. To test their potency to up-regulate APOA1 transcript levels, these oligonucleotides (termed AntagoNATs) were first tested in HepG2 cells. Forty-eight hours after transfection, total RNA was isolated from these cells and APOA1 mRNA concentration was measured by quantitative RT-PCR. A number of AntagoNATs induced a significant increase of APOA1 mRNA expression (Figure S4). These active AntagoNATs appeared to cluster into two major “hot spots” on the APOA1 NAT sequence. To confirm the existence of the hot spots we then chose two regions for a more detailed survey in single nucleotide steps: one around a hot spot (CUR-0461 to CUR-0284), the other – around a mostly inactive area (CUR-0279 to CUR-0473). AntagoNATs designed around the hot spot produced on average a 1.7 fold up-regulation of APOA1 mRNA, while oligonucleotides designed around an inactive area produced an up-regulation of 1.05 fold on average, which supports the hot spot hypothesis (Figure S4). We then mapped active oligonucleotides to the secondary structure of APOA1 NAT. Active AntagoNATs were associated almost exclusively with one arm of the molecule. The two “hot spots”, which are separated by about 200 bases in the linear NAT sequence, mapped close to each other in the secondary structure and were associated with a system of stable hairpin loops. These results indicate that differences in AntagoNAT activity may be due to their interactions with secondary and consequently tertiary structure of NATs. Finally, AntagoNATs invoking the largest up-regulation were selected and chemically modified to increase their stability. They were then tested in vitro, in both in human (HepG2) and African green monkey cells, to identify the combination of sequence and chemical modifications that produced the highest up-regulation of APOA1 in both species. For this transition between human and monkey cells, the AntagoNATs were specifically designed based on regions for which the human and rhesus genome sequences were identical. Figure 5 demonstrates the increase in APOA1 expression observed in HepG2 cells following treatment with two such AntagoNATs, the most active of which (CUR-1906) induced a 2–4-fold up-regulation of APOA1 mRNA and protein levels (see also Figure S5). Figure 6A demonstrates APOA1 RNA expression in primary monkey hepatocytes treated with active AntagoNATs relative to a same-chemistry control oligonucleotide (broken line). The sequences and modifications of these AntagoNATs, as well as the regions targeted by each, are presented in figure S6. The majority of the chemically modified AntagoNATs screened induced no cytotoxicity at concentrations up to 4000nM, as determined by the MTS test. From this pool of active AntagoNATs, CUR-962, a 12-mer single-stranded oligonucleotide with phosphorothioate backbone and 5 LNA modifications in gapmer configuration (LLXXXXXXXLLL where L=LNA), was selected and manufactured on a larger scale for in vivo testing.

Figure 5. Antisense oligonucleotides targeting APOA1-AS can increase APOA1 mRNA and protein expression in vitro.

Figure 5

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HepG2 cells were transiently transfected with AntagoNATs against APOA1-AS. CUR-1906 induced a 2–4 fold up-regulation of APOA1 mRNA (A) and protein (B) levels (3<n<6; p<0.05) (Graphs represent mean relative expression values ± SEM; * = p<0.05)

Figure 6. Antisense oligonucleotides targeting APOA1-AS can increase APOA1 mRNA and protein expression in vivo.

Figure 6

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A) Primary African green monkey hepatocytes were transiently transfected with AntagoNATs against APOA1-AS and induced a 2–4 fold up-regulation of APOA1 mRNA. (B) Liver biopsies of monkeys (n=4) treated with CUR-962 showed ~1.7 fold increase in APOA1 mRNA concentration compared to baseline biopsy samples (P=0.04). All four active AntagoNAT treated monkeys showed elevated liver APOA1 mRNA concentration. (C) Measurement of APOA1 protein in African green monkey blood (n=4). We observed significant increase in circulating APOA-1 protein by an average of 10 mg/dl and up to 15 mg/dl. (All graphs represent mean values ± SEM; * = p<0.05, ** = p<0.01, *** = p<0.001).

African green monkeys (N=4/group) received three 10 mg/kg intravenous injections of the AntagoNAT (CUR-962), or a chemically matched inactive control AntagoNAT (CUR-963), over a five days period (days 1, 3 and 5). The CUR-963 control was seen to have no affect on APOA1 mRNA or protein expression compared to baseline values during prior in-vitro testing. RT-PCR analyses of liver biopsies taken three months prior to injection and 72 hours after final injection, revealed a statistically significant increase in APOA1 mRNA expression for all four monkeys that received the active AntagoNAT, CUR-962, compared to those receiving the control CUR-963. On average, liver biopsies from the AntagoNAT-treated cohort demonstrated ~1.7 fold intra-individual increase of APOA1 mRNA compared to pre-treatment biopsy samples (P=0.04; Figure 6B), while expression levels in the control CUR-963-treated group were unchanged. Furthermore circulating APOA1 protein concentrations in the AntagoNAT-treated animals were elevated by ~10–15 mg/dL on days 6, 15 and 20 post-injection, compared to their matched pre-treatment biopsies (P<0.01; Figure 6C).

Discussion

Within the last decade, non-coding RNAs have come to represent an exciting new avenue for the treatment of diseases (Calin and Croce, 2006; McDermott et al., 2011; Miller and Wahlestedt, 2010; Pastori and Wahlestedt, 2012; Taft et al., 2010; van Rooij and Olson, 2007). By identifying and characterizing novel regulatory antisense transcripts, it may be possible to manipulate the expression of a therapeutic gene for a range of indications. In this regard, siRNAs, which directs selective mRNA degradation using the multi-component RNA-induced silencing complex (Zamore et al., 2000), offers a fast and robust approach to study RNA-mediated gene regulatory mechanisms in vitro. Alternatively, short antisense oligonucleotides can be specifically modified for increased stability, high specificity and potency, and are a powerful tool to attain direct gene silencing in vivo (Dias and Stein, 2002; Veedu and Wengel, 2009). Here we have identified a mechanism by which an endogenous, long non-coding antisense RNA can modulate the expression of APOA1 and multiple neighboring genes, by facilitating the interaction of histone–modifying complexes with a specific target locus. ChIP analyses of H3K4, H3K9 and H3K27 tri-methylation status following APOA1-AS knockdown demonstrated that the interplay between H3K27 methylation and H3K4 demethylation within the same chromatin region is critical to maintaining an active chromatin state for APOA1, APOC3 and APOA4 expression.

LSD1 is known to remove methyl marks from H3K4 primarily, which are associated with transcriptionally active promoters, and pharmacological LSD1 inhibitors have also been shown to enhance H3K4 methylation leading to the de-repression of epigenetically suppressed genes (Wang et al., 2011a). Though it has been reported that LSD1 is only effective at removing methyl groups from mono- or dimethylated H3K4 (Shi et al., 2004), it is capable of binding H3 peptides with trimethylated K4 residues (Stavropoulos et al., 2006) and knock-down of LSD1 has previously also been shown to increase levels of H3K4 trimethylation at target genes (Adamo et al., 2011). As such, while LSD1 may not be directly affecting the trimethylated form of H3K4 under normal conditions, it is possible that its removal may provide a more optimum environment for histone methylation. The long intergenic RNA (lincRNA) HOTAIR has previously been shown to act as a modular scaffold by binding to and recruiting LSD1 to its chromatin targets and thus silencing gene expression (Tsai et al., 2010b). Our data suggests that the APOA1-AS transcript identified here induces epigenetic regulation of APOA1 in a similar manner, by mediating LSD1 occupancy across this locus. Treatment with our APOA1-AS siRNAs led to the disruption of this interaction, which coincided with an increase in H3K4 trimethylation within the region encompassing APOA1 (primer sets 1 to 4), thus resulting in enhanced gene expression. This would appear to be H3K4-specific as the APOA1-AS siRNA was not seen to affect the trimethylation state of H3K9, which is in line with previous reports that LSD1 demonstrates an extremely high affinity for H3K4 over H3K9 (Shi et al., 2004).

Similarly, various other lncRNAs have also recently been reported to physically interact with the PRC2 component SUZ12. For example, the NAT ANRIL mediates the silencing of tumor suppressor gene p15INK4B through physical recruitment of SUZ12, which in turn induces repressive H3K27 trimethylation (Kotake et al., 2011). Indeed, in addition to binding LSD1, HOTAIR has also been shown to repress the transcription of the human HOXD locus through its direct interaction with SUZ12, and thus the PRC2 complex (Tsai et al., 2010b). Here we demonstrate that knockdown of APOA1-AS leads to disruption of SUZ12 binding across the majority of the APO gene cluster and coincides with a reduction of H3K27 trimethylation marks along the promoter regions of APOA1, APOA4 and APOC3. While this is likely to also promote APOA1 gene expression, along with decreases in LSD1, a reduced occupancy of SUZ12 would also explain the observed increase in APOA4 and APOC3 expression in liver cells following APOA1-AS siRNA treatment.

Interestingly, no trans- effect was indicated for the APOA1-AS on APOA5, which is located 28kb downstream of the other three affected genes, nor was there any effect on the neighboring upstream gene QSK, which may indicate a boundary for the NAT’s regulatory effect on the chromatin or the presence of insulator elements (Kim et al., 2007). Indeed, a previous study has shown that Apo/A1/C3/A4 genes are organized into complex chromatin loops (Mishiro et al., 2009), and QSK and APOA5 are not present in the loop.

The locus-specific recruitment of histone modifying complexes by NATs, observed here and in our other published report (Modarresi et al., 2012), suggests that approaches to modify these antisense transcripts may offer more control over target sense gene up-regulation than compounds, such as LSD1 or PRC2 inhibitors, which could have a number of off-target effects. It should be noted that the use of ASOs to mediate RNA silencing can also be associated with off-target activity, however the fact that the same effect of APOA1 up-regulation was observed following treatment with both siRNAs and modified AntagoNATs supports the specificity of the mechanism proposed here. Furthermore, the up-regulation of APOA1 observed during AntagoNAT screening and in vivo testing are unlikely attributed to general toxicity, as indicated by MTS toxicity assays, nor could it be an artifact of PS/2OMe/LNA/DNA chemistry load, as much higher concentrations of the relevant control AntagoNATs were seen to have no effects. To our knowledge, the present data, showing the effectiveness of a relatively low dose of CUR-962 in increasing APOA1 mRNA and protein concentration in vivo in a primate model, highlights the potential applicability of AntagoNAT strategies for inducing therapeutic gene up-regulation in a clinical setting. Although still in its infancy, further optimizing this approach could have advantages over current drug-based or viral gene therapy approaches due to the high specificity, stability, and potency of antisense oligonucleotides.

Experimental Procedures

Cell culture

All cell lines were maintained at 37°C and 5% CO2 and passaged every 3–4 days. FBS from Mediatech (cat# MT35-011-CV) was used in all cases. 518A2 and Vero76 cells were grown in DMEM +5% FBS. HepG2 cells were grown in EMEM (ATCC cat #2003) +10% FBS. African green monkey primary hepatocytes were grown in DMEM and isolated as previously reported (LeCluyse et al. Methods Mol Biol 2005, 290:207-29). All media contained penicillin/streptomycin (Mediatech cat# MT30-002-CI).

RACE

Rapid amplification of the 3′ or 5′ cDNA ends (RACE) was conducted using the First Choice RLM RACE kit (Life Technologies cat#AM1700) as described by the manufacturer, followed by two successive nested PCRs of the cDNA copies. The PCR products were cloned and sequenced by Davis Sequencing (Davis, CA).

Transient transfections

For each transfection of active siRNA and negative control siRNA, 400 ul of OptiMEM was mixed gently with 4 ul of Lipofectamine 2000 (Invitrogen), 20 ng of active/negative siRNA was added to the above mixture and incubated for 20 minutes at room temperature. After 20 minutes, 400 ul of OptiMEM+Lipofectamine+siRNA mixture was added to the HepG2 cells (2×105cells/well) that were seeded into six-well plates (35-mm) in EMEM (ATCC cat #2003) +10% FBS. Cells were maintained at 37 °C and 5% CO2. After 24 hours, media was replaced with fresh EMEM +10% FBS. 48 hours after transfection, cells were collected and the RNA was extracted using Qiagen RNA extraction column according to manufacturer’s protocol.

RNA extraction

Total RNA was isolated using Qiagen Midi RNA Extraction kit (Qiagen) or SV Total RNA Isolation System from Promega (cat # Z3105) following the manufacturers’ instructions.

Reverse transcription and quantitative PCR

Reverse transcription reaction was performed using High Capacity cDNA kit from Applied Biosystems (cat#4368813) as described in the manufacturer’s protocol. Real time PCR was conducted using ABI Taqman Gene Expression Mix (cat#4369510) and probes designed by ABI (assay ID#Hs00202021_m1 for APOA1) on the StepOne Plus Real Time PCR system (Applied Biosystems). The data was normalized to 18S expression (ABI, cat# 4319413E).

Chromatin immuno-precipitation assay

HepG2 cells were grown in EMEM (ATCC cat #2003) +10% FBS and transiently transfected with 20 ng of control and APOA1 according to the transient transfection protocol explained above. 48 hours after transfection, ~ 2×107 cells were trypsinized, collected and washed with ice-cold PBS three times and the cell pellet was resuspended in cell lysis buffer (85 mM KCl, 0.5% Nonidet P-40, 5 mM HEPES, pH 8.0) supplemented with protease inhibitor cocktail (Roche), incubated on ice for 15 min, and centrifuged at 3,500 g for 5 min to pellet the nuclei. The pellet was resuspended in nuclear lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA pH 8.0, 1% Triton X-100 0.1% Sodium Deoxycholate, 0.1% SDS) at a ratio 2:1 (v/v) relative to the initial cell pellet volume and incubated on ice for 10 min. The solution was sonicated to obtain chromatin fragments of size 100bp-1000 bp using bioruptor for 10 minutes with 30 sec on and 30 sec off cycles. The sonicated lysate was centrifuged at 13,000 rpm for 5 min at 4°C and the supernatant was aliquoted for each ChIP reaction including one aliquot as input.

Each aliquot was diluted to 10 times with RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA pH 8.0, 1% NP-40, 0.5% Sodium Deoxycholate, 0.1% SDS) supplemented with protease inhibitor cocktail (Roche) and PMSF. For H3K4me3, H3K9me3 and H3K27me3 experiments, 5 ug of antibody, including IgG as a control, was added to each ChIP and incubated at 4°C for 4 h with rotation. 20 μl of protein A/G beads mixture (pre-adsorbed with sonicated single stranded herring sperm DNA and BSA for 30 min at room temperature) was added to all samples and IP for two hours at 4°C with rotation at. For Suz12 and LSD1 experiment s, 8 ug of antibody, including IgG as control, was incubated with 50 μl G-protein Dynabeads (Life Technologies) for 30 min at room temperature. This antibody/Dynabeads mixture was added to all samples and IP, and incubated overnight at 4°C with rotation.

The beads were collected using a magnetic rack (Invitrogen) and the supernatant was discarded. The beads were washed with 1 ml of the buffer in the following order: 3X with low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA pH 8.0, 150 mM NaCl, 20 mM Tris-HCl pH 8.0), 1X with high salt buffer (0.1% SDS,1% Triton X-100, 2 mM EDTA pH 8.0, 500 mM NaCl, 20 mM Tris-HCl pH 8.0) and 2X with TE buffer, each time washing the beads with rotation for 5 min at 4°C and discarding the supernatant. The beads were then incubated with 200 ul of DNA elution buffer (1% SDS, 100 mM NaHCO3) at room temperature and the supernatant was collected. DNA was purified from the eluate using Qiagen DNA mini kit following manufactures protocol.

Primate Studies

In vivo studies were conducted at the St. Kitts Biomedical Research Foundation, St. Kitts, West Indies, in full compliance with NIH Guide for the Care and Use of Animals. Baseline clinical exams, including clinical chemistries, were conducted on 8 adult female African green monkeys to confirm good health and suitability for study enrollment. Monkeys were fed approximately 120 g per day standard monkey chow (TekLad, Madison, WI). The monkeys were assigned to 2 treatment groups of 4 animals each and dosed once daily between 7:00 and 10:00 a.m on study days 1, 3, and 5 by intravenous saphenous vein infusion over ~15–20 min at a rate of 24 ml/kg/h. The animals were sedated with ketamine and xylazine IM (5.0 mg/kg ketamine/1.0 mg/kg xylazine) prior to and during the dosing procedure. Blood samples were obtained via superficial venipuncture from all animals at three baseline time points prior to treatment. Additional blood samples were collected at intervals post-dosing and plasma APOA1 protein concentrations were assessed by immunoturbidometric assay. All sampling was performed prior to feeding after 12 hours without access to food to minimize dietary effects APOA1 measurements. A percutaneous liver biopsy was performed on all study monkeys under ketamine and xylazine sedation at the first baseline sampling time point and on study day 7 employing an INRAD 14 gauge biopsy needle to obtain 4 core biopsies (~1.5 cm in length) from the right lobe of the liver. Biopsies were immediately immersed in a labeled cryotube containing 2 ml of RNAlater (Qiagen) and incubated at 4°C overnight, following which the RNAlater was aspirated and the sample tube flash frozen in liquid nitrogen for transportation prior to total RNA isolation for real-time qPCR.

Supplementary Material

01

Highlights.

  • An endogenously expressed natural antisense transcript was characterized for APOA1

  • APOA1-Antisense (APOA1-AS) acts as a negative regulator of APOA1 transcription

  • APOA1-AS silences target gene expression by recruiting histone-modifying enzymes

  • Inhibiting APOA1-AS can increase APOA1 expression in vitro and in vivo

Footnotes

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Supplementary Materials

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