Periodic Reporting for period 1 - MIR-CHROM-C (Investigating the microRNA-chromatin remodelling circuitry in cardiac development)
Okres sprawozdawczy: 2016-04-01 do 2018-03-31
Future treatments of cardiovascular conditions will benefit from our ability to harness progenitor cells or stem cells for therapeutic purpose. To achieve this promising goal the objective of this Marie-SkŁodowska-Curie Action is to dissect the molecular mechanisms that govern fundamental cellular differentiation processes in their normal physiological setting, the developing embryo. One such fundamental process is the control of cell lineage determination. This is important during both stem cell differentiation and embryo development, including heart development. In the heart, many of the diffusible signalling molecules, transcription factors and more recently non-coding RNAs and epigenetic factors that contribute to this process have been identified. This has facilitated rapid advances in our understanding of the molecular mechanisms underlying the control of cell fate choice, however many details remain to be elucidated. This Action will focus on enzyme complexes that remodel chromatin during the epigenetic changes that occur during heart development. These complexes, which consist of different BAF-subunits and a core enzyme called Brg1, determine whether or not chromatin is accessible to transcriptional regulators, and they form an important nexus governing lineage decisions. The Action will address an important gap in our understanding concerning the regulation of subunit composition of the complexes and how subunit composition affects their function. This project builds on recent pilot data and will use in vivo methods to investigate the regulation of chromatin remodelling factors by microRNAs during cardiac development in experimentally accessible chick embryos. Mechanistic gain- and loss-of-function experiments in embryos will be complemented by genomic approaches to determine the genome-wide dynamic coverage of BAF/Brg1 complexes during heart development.
After some months working in the Münsterberg lab, I successfully established approaches to interfere with microRNA function in vivo, in developing heart of chick embryos, similar to approaches previously published in somites. Briefly, miR function can be inhibited effectively by microinjection of fluorescently tagged and highly specific antagomirs into the early heart tube. Following incubation for 24-72hrs, localization of FI-antagomir can be visualized and the knock down (KD) or double knock down (DKD) of miR expression confirmed by qPCR. This project will examine heart phenotypes resulting from miR DKD. (Panel A)
To determine whether this interaction is also important in cardiac development, we examined BAF60a/b expression after double-knockdown of miR-1 and miR-133 function using targeted microinjection of specific antagomirs. qPCR analysis showed reduced expression of both miRs after DKD, however, we found no significant derepression of the miR-133/miR-1 targets BAF60a and BAF60b. This suggests that interactions of miRs with their respective target genes are highly tissued specific; we hypothesise that negative regulation of the BAF60 variants in the heart requires additional miRs. Overall our observations were consistent with microarray analysis in mice, where the genetic double knock-out of miR-1and miR-133 had no significant effect on BAF60a/b expression.
To assess the efficacy of antagomir-mediated DKD in chick hearts, we examined the expression of genes reported to be affected in the genetic double knock-out. This confirmed a similar profile in chick as was seen in mouse: increased expression of Kcnmb1 and Myocardin, targets for miR-133 and miR-1 respectively, no change for Ccnd2 or Kcnd2, and negative effects on Eya1 and Six1 transcription factors - a secondary consequence of loss of microRNA function, as they are no direct targets of miR-1 or miR-133. Direct targets are expected to be relatively increased, or ‘derepressed’, in absence of miR function. (Panel B)
To validate multiple predicted miR:target gene interactions I used an established pipeline and perform luciferase assays and this has been complemented by in vivo experiments using antagomirs. Using our DKD approach we will systematically inhibit miRs predicted to target components of the BAF/Brg/Brm complex. It is known that BAF60b is downregulated in cardiomyocytes. However, inhibition of miR-1 did not prevent this negative regulation. We hypothesised that this may be due to redundancy with miR-130, which is highly expressed in the heart and we already confirmed interacts with the 3’UTR of BAF60b (Smarcd2) (unpublished), and moreover, miR-19 can play an important role in this modulation because Baf60a and Baf60b are both predicted targets. Thus, we anticipated that DKD of miR-1/miR-130 will lead to derepression of BAF60b expression. Similarly, BAF60a is a predicted target for miR-101, which we showed by small RNA profiling is enriched in the developing heart. We will perform DKD for miR-133 and miR-101 and examine effects on BAF60a expression. In addition, miR-101 is predicted to target Brg1, which we found was derepressed after inhibition of miR-1. Previous small RNA profiling identified, miR-19, miR-101 and miR-130 as highly enriched during cardiogenesis (unpublished). (Panel C)
Embryos between HH stage 14 and 15 have been used and phenotypes have been examined after 24 hours of incubation, and I observed that the survival rate was smaller on DKO1 embryos in comparison with SCR, whereas in the other two conditions was not significant. Moreover, I analyzed the heart rate in these embryos and I could observe that only DKO1 showed a slow heart rate that could imply a bradycardia but the other injected embryos were normal. (Panel D)
To determine whether this interaction is also important in cardiac development, we examined BAF60a/b expression after double-knockdown of miR-1 and miR-133 function using targeted microinjection of specific antagomirs. qPCR analysis showed reduced expression of both miRs after DKD, however, we found no significant derepression of the miR-133/miR-1 targets BAF60a and BAF60b. This suggests that interactions of miRs with their respective target genes are highly tissued specific; we hypothesise that negative regulation of the BAF60 variants in the heart requires additional miRs. Overall our observations were consistent with microarray analysis in mice, where the genetic double knock-out of miR-1and miR-133 had no significant effect on BAF60a/b expression.
To assess the efficacy of antagomir-mediated DKD in chick hearts, we examined the expression of genes reported to be affected in the genetic double knock-out. This confirmed a similar profile in chick as was seen in mouse: increased expression of Kcnmb1 and Myocardin, targets for miR-133 and miR-1 respectively, no change for Ccnd2 or Kcnd2, and negative effects on Eya1 and Six1 transcription factors - a secondary consequence of loss of microRNA function, as they are no direct targets of miR-1 or miR-133. Direct targets are expected to be relatively increased, or ‘derepressed’, in absence of miR function. (Panel B)
To validate multiple predicted miR:target gene interactions I used an established pipeline and perform luciferase assays and this has been complemented by in vivo experiments using antagomirs. Using our DKD approach we will systematically inhibit miRs predicted to target components of the BAF/Brg/Brm complex. It is known that BAF60b is downregulated in cardiomyocytes. However, inhibition of miR-1 did not prevent this negative regulation. We hypothesised that this may be due to redundancy with miR-130, which is highly expressed in the heart and we already confirmed interacts with the 3’UTR of BAF60b (Smarcd2) (unpublished), and moreover, miR-19 can play an important role in this modulation because Baf60a and Baf60b are both predicted targets. Thus, we anticipated that DKD of miR-1/miR-130 will lead to derepression of BAF60b expression. Similarly, BAF60a is a predicted target for miR-101, which we showed by small RNA profiling is enriched in the developing heart. We will perform DKD for miR-133 and miR-101 and examine effects on BAF60a expression. In addition, miR-101 is predicted to target Brg1, which we found was derepressed after inhibition of miR-1. Previous small RNA profiling identified, miR-19, miR-101 and miR-130 as highly enriched during cardiogenesis (unpublished). (Panel C)
Embryos between HH stage 14 and 15 have been used and phenotypes have been examined after 24 hours of incubation, and I observed that the survival rate was smaller on DKO1 embryos in comparison with SCR, whereas in the other two conditions was not significant. Moreover, I analyzed the heart rate in these embryos and I could observe that only DKO1 showed a slow heart rate that could imply a bradycardia but the other injected embryos were normal. (Panel D)
Treatment of cardiovascular conditions will benefit from the use stem cells for therapeutic purpose. To help achieve this, we will dissect the mechanisms underlying cell differentiation in development, as similar factors are important in embryos and stem cells. Many signals and molecular regulators contributing to heart development have been identified. However, details regarding the mechanisms underlying cell differentiation remain to be elucidated. This project will focus on complexes that change chromatin, which packages DNA inside the cells’ nucleus. These contain core enzymes and different associated factors (BAFs), which determine whether chromatin, and therefore the genes encoded in DNA, is accessible to regulators. Thus, the complexes are controlling cardiac development. We have found that small RNAs, called microRNAs, determine the composition of the complexes by regulating some of the associated factors: BAFs. We will inhibit the function of microRNAs and study the consequences for complex composition and heart development. This will use state-of the art in vivo methods and provide important mechanistic understanding of cell differentiation processes.
The outcome will be a better understanding of molecular mechanisms governing fundamental cellular processes, such as cell fate specification within the embryo. This will benefit the design of effective approaches to repair a defective heart by using stem cells.
The outcome will be a better understanding of molecular mechanisms governing fundamental cellular processes, such as cell fate specification within the embryo. This will benefit the design of effective approaches to repair a defective heart by using stem cells.