by Jong Won Lim, postdoctoral fellow
Mentors: Galina Filippova, PhD and Stephen Tapscott, MD, PhD
See grant Targeting Epigenetic Silencing of the 4qD4Z4 repeat array in FSHD
A. SPECIFIC AIMS:
are not changed from the original application.
B. STUDIES AND RESULTS:
Aim 1. Determine whether D4Z4-derived endogenous small RNAs mediate DUX4 epigenetic silencing
Facioscapulohumeral muscular dystrophy (FSHD) is caused by incomplete repression of the D4Z4 repeat array on the disease-permissive chromosome 4q resulting in aberrant expression of DUX4, the candidate FSHD gene imbedded within the D4Z4 repeat. We and others identified multiple sense and antisense long noncoding transcripts and small RNAs derived from the 4qD4Z4 repeat array. Considering that small RNAs have been implicated in Argonaute (Ago)-mediated heterochromatin formation at repetitive sequences and that bidirectional transcripts from D4Z4 may form double stranded RNAs for small RNA processing, we have designed several siRNAs corresponding to the endogenous D4Z4 small RNAs and shown that siRNAs targeting the DUX4 promoter region increased repressive histone marks H3K9me2 and efficiently silenced DUX4. These data suggested that endogenous 4qD4Z4/DUX4 noncoding RNAs may epigenetically regulate 4qD4Z4 repeats via Ago/RNA-mediated recruitment of histone methyltransferases (HMTs) leading to DUX4 transcriptional silencing.
Validation of Ago-1, Ago-2 and Dicer1 siRNAs
Figure 1. Validation of Ago2 and Dicer1 siRNAs in FSHD2 cell line. Ago-2 siRNAs (Ago2 N01-N03) and Dicer1 siRNAs (Dicer1 N01-N03) were transfected twice (second transfection after 2 days) into FSHD2 cell line. Total RNA samples were prepared at 5 days post-transfection. Expression of Ago-2 (a) and Dicer1 (B) were analyzed by Q-RT-PCR.
To test whether components of the small RNA processing machinery including Argonautes (Ago-1 or Ago-2) and Dicer1 are required for epigenetic silencing of D4Z4/DUX4, we have designed and validated Ago-1, Ago-2, and Dicer1 siRNAs. Since it has been challenging to knockdown these RNA processing enzymes using siRNA pathway, in addition to newly designed siRNAs, we tested Ago-1 and Ago-2 antisense oligos/gapmers that utilize RNase H pathway to target specific transcripts (ISIS pharmaceuticals). We used a double transfection protocol to achieve efficient and prolonged knock down. At least two Ago-2 and two Dicer1 siRNAs showed ~70 – 80% knockdown (Fig 1). In addition both Ago-1 and Ago-2 gapmers resulted in significant knock down of Argonautes (Ago-1 gapmer data not shown). We are currently using these siRNAs and gapmers on control lines (NR-135, 2397, and 2401) with relatively short D4Z4 array length. If Argonautes are indeed involved in DUX4 silencing in non-pathogenic condition, we expect that knockdown of Ago-1, Ago-2, or Dicer1 will reactivate DUX4 expression and change D4Z4 chromatin state in control cell lines similar to SMCHD1 knockdown.
Aim 2. Determine whether small RNAs targeting the DUX4 promoter region can induce epigenetic silencing of DUX4
Previous reports showed that small RNAs targeting promoter of a gene can silence the gene through Argonaute and RNA mediated gene silencing process. Long noncoding transcripts associated with the promoter region were also shown to be involved in the siRNA-mediated epigenetic silencing (Hawkins et al.2009; Kim et al., 2006). We have previously reported that several small RNAs targeting promoter of the DUX4 gene decreased expression of DUX4 and DUX4 target genes and increased levels of H3K9me2, a repressive histone mark, at the DUX4 promoter region. Using publically available small RNA sequencing datasets (Ameyar-Zazoua et al., 2012), we found that some small RNAs derived from D4Z4 repeats are associated with chromatin and form clusters of small RNAs (see Fig 2). To confirm biological functions of these chromatin associated small RNAs, we are currently testing whether exogenous siRNAs targeting these ‘cluster’ regions can silence DUX4 and whether sense or antisense noncoding D4Z4 transcripts are required for DUX4 silencing by our siRNAs.
Test new DUX4 siRNAs
Figure 2 Test new DUX4 siRNAs in FSHD2 cell line. (A) The diagram shows structure of the DUX4 gene and target location for new DUX4 siRNAs targeting clustered regions of chromatin-associated D4Z4 small RNAs (shown as short vertical black lines). ASPC-S, sp01 and sp02 siRNAs target the sense noncoding transcripts associated with D4Z4 and ASPC-AS, sp01-AS and sp02-AS siRNAs target the antisense transcripts. (B) DUX4 siRNAs were transfected into FSHD2 myoblasts. Total RNA was prepared at 4 days post-transfection time point. Expression of DUX4 was analyzed by Q-RT-PCR. DUX4 siRNA. * sc03 is a DUX4 codingregion siRNA used as a positive control.
We have designed new DUX4 siRNAs targeting either sense (S) or antisense (AS) transcripts at the upstream (5’-end) clustered region of the chromatin associated endogenous small RNAs and at the DUX4 promoter region and tested whether these siRNAs might affect DUX4 expression in FSHD2 myoblasts by using siRNA transfection and Q-RT-PCR (see Fig 2). One of the newly designed DUX4 siRNAs, ASPC-AS, targeting an antisense transcript at the clustered region, significantly decreased DUX4 expression. Interestingly, for the promoter region only siRNAs targeting sense strand (sp01 and sp02) worked for DUX4 knockdown. These data suggest that in addition to the promoter-associated small RNAs, the endogenous chromatin-associated small RNAs at the upstream ‘cluster’ region may also play a role in DUX4 epigenetic silencing. Moreover, different long non-coding D4Z4 transcripts (antisense at the upstream ‘cluster’ region and sense at the DUX4 promoter region) may be involved in this process. We will examine whether these D4Z4 transcripts are required for our exogenous DUX4 siRNAs’ function by using strand specific RT-PCR and antisense oligonucleotides (ASO/gapmers) targeting either sense or antisense D4Z4 transcripts via RNAse H pathway.
Test the DUX4 promoter siRNAs in FSHD1 cell line
We have previously shown that several exogenous siRNAs targeting non-coding D4Z4 regions can efficiently silence DUX4 in FSHD2 myoblasts. To test whether similar mechanisms apply to FSHD1 cells, we tested our siRNAs in FSHD1 myoblasts (FSHD-183). Although our preliminary data suggest that our siRNAs work in FSHD1 cells, the results were inconclusive due to low DUX4 expression levels in FSHD183 cells. We have tested several additional FSHD1 lines for DUX4 expression and will confirm function of our DUX4 siRNAs in these lines (e.g. 54-2).
Generate DUX4 promoter-luciferase reporter C2C12 stable cell lines
According to the RNA-mediated transcriptional gene silencing model, small RNAs targeting a promoter region may bind either directly to chromatin or to a transcript associated with the promoter region of the target gene. To test whether our DUX4 promoter siRNAs can silence the DUX4 promoter without additional DUX4 coding sequences, we are currently generating C2C12 stable lines with stably integrated DUX4 promoter-luciferase reporters. After validating the DUX4 promoter-luciferase stable lines, we will transfect our DUX4 promoter siRNAs into these lines to test whether our DUX4 siRNAs can decrease expression of the reporter gene.
Co-transfection of the DUX4 promoter siRNAs with Ago-1/Ago-2 siRNAs
Previous reports suggested that Argonaute proteins (Ago-1 or Ago-2) are the key components of transcriptional gene silencing (TSC) complex that induces epigenetic changes after recruitment to the promoter region by small RNAs. Using the validated Ago-1 and Ago-2 siRNAs and gapmers (see Aim 1, Fig.1), we are examining whether function of our DUX4 promoter siRNAs requires Argonaute activities by cotransfecting both DUX4 siRNAs and Ago siRNAs into FSHD2 cell line. If our DUX4 siRNAs downregulate the DUX4 gene through Ago-associated complex, knockdown of Ago will block DUX4 silencing. We will also test whether our DUX4 siRNAs (Fig. 2) down regulate the DUX4 gene through epigenetic changes via transcriptional gene silencing (TGS) rather than post-transcriptionally through RNAimediated transcript degradation by using DUX4 ASO/gapmers that utilize the RNaseH pathway to target either sense or antisense D4Z4 transcripts.
This project will provide new insights into the mechanisms of epigenetic silencing of the D4Z4 array in non-pathogenic conditions and will identify efficient therapeutic targets inducing long-term epigenetic silencing of DUX4. We will define key regulators involved in D4Z4/DUX4 silencing and depending on the epigenetic mechanism identified (transcriptional gene silencing vs posttranscriptional gene silencing), we will develop efficient therapeutic applications: single or double stranded siRNAs vs antisense oligonucleotides.
D. FUTURE PLANS:
Next 6 months:
1) We will complete testing whether Ago-1, Ago-2, and Dicer1 are required for endogenous D4Z4 epigenetic silencing (aim 1) and by our exogenous DUX4 siRNAs (aim 2)
2) We will complete generation and validation of the DUX4 promoter-reporter stable lines (aim 2)
3) We will perform strand-specific RT-PCR to test whether our DUX4 promoter siRNAs may target noncoding transcription through RNAi pathway and to validate DUX4 ASO/gapmers (aim 2).
4) We will test biological functions of the endogenous small RNAs by knocking down sense or antisense D4Z4 transcripts via RNaseH pathway by using antisense oligonucleotides (ASO/gapmers) in collaboration with ISIS and Lauren Snider (Tapscott lab). Using these ASOs, we will also test whether our exogenous DUX4 siRNAs require sense or antisense transcripts for DUX4 silencing (aim 1 and 2).
In the Future:
1) Based on small RNA deep sequencing data from control and FSHD myoblasts/myotubes (suppl. Proposal/ a separate progress report will be prepared), we will design new DUX4 siRNAs that may mimic endogenous small RNAs and test whether these siRNAs can affect DUX4 expression and chromatin state.
2) If function of our DUX4 siRNAs is Ago-dependent, we will test whether DUX4 siRNA transfection may recruit Ago complex to D4Z4 (noncoding) transcripts and induce association of siRNA/Ago complex/D4Z4 transcripts with repressive chromatin by using Ago- and H3K9me2- RIP-seq.