Profiling The Epigenetic Interplay Of RUNXOR And RUNX1 In Breast Cancer Cells By LncRNA In Situ Activation

Background:RUNX1(Runt related transcription factor 1)is a master regulator in definitive hematopoiesis,and aberration of which is frequently involved in a variety of hematological malignancies including acute myeloid leukemia.Recently,due to the great advances of next-generation sequencing,accumulating evidences have suggested that RUNX1 may also play a major role in breast cancers.In 2012,two research groups independently reported that RUNX1 was one of the most common mutations/deletions found in breast cancer.Later,it was shown that RUNX1 gene had a special temporal/spatial expression pattern throughout the different phases of mammary gland development,indicating its functional role in this tissue.Besides,RUNX1 is also involved in the fate determination of mammary epithelial cells by regulating lineage differentiation and stem/progenitor cell potential.However,the evidence to date shows that the RUNX1 gene can have dualistic roles with regard to promoting or suppressing breast cancer phenotypes,and that this may be aligned to different subtypes of the clinical disease.The exquisite control of RUNX1 expression is the fundamental basis of RUNX1 performing its normal function.At the transcriptional level,RUNX1 can be transcribed from two alternative promoters,including a distal promoter P1 and a proximal promoter P2.Activation of different promoters can give rise to RUNX1 isoforms with varied amino termini,primarily RUNXlc and RUNXlb,although few functional discrepancies have been found between these two.The promoters are independently activated in selective tissues in a spatial/temporal dependent manner.Alternative promoter usage within the same gene is a general phenomenon in gene expression,which guarantees that a proper dosage of RUNX1 will be transcribed at any given time.So far,the mechanisms controlling alternative promoter usage of the RUNX1 gene remained unknown.Long noncoding RNAs represent a key component of gene expression regulators.It s involved in basically every step of gene expression regulation,and plays a pivotal role in controlling transcription,post-transcriptional modification and translation,etc.Our lab has recently identified a long noncoding RNA RUNXOR(RUNX1 overlapping long noncoding RNA,RUNXOR)within the RUNX1 locus,and further confirmed that the 3' fragment of IncRNA RUNXOR can interact with multiple cis-regulating elements within the RUNX1 locus,including RUNX1 promoter and enhancer,indicating its potential regulatory role.Nonetheless,as a 260kb unspliced IncRNA,RUNXOR is very hard to be studied using conventional vectors.Thus far,the exact functions of RUNXOR remain elusive.For example,if and how may IncRNA RUNXOR regulate its neighboring gene RUNX1;further,what's the possible role of RUNXOR in malignancies where RUNX1 dysregulation frequently occurs.Deciphering these mysteries is important for improving our understandings of the mechanisms behind RUNX1 regulation and possibly deregulation in tumors,which may serve as a potential therapeutic target for breast cancers patients.Objective:To investigate the potential regulatory impact of IncRNA RUNXOR on its neighboring gene RUNX1 and the underlying mechanisms behind RUNX1 deregulation.The effects of RUNXOR-RUNX1 axis activation on the malignant phenotype of breast cancer are also explored.Methods:Pearson correlation analysis was initially performed to study the potential relationship between IncRNA RUNXOR and RUNX1 mRNA.We then devised a novel CRISPR-Cas9 mediated "endogenous IncRNA in situ activation" approach to activate the endogenous RUNXOR gene by inserting a potent CMV promoter in front of RUNXOR.This model was then used to further explore the effect of RUNXOR on the regulation of RUNX1 transcription and potentially its alternative promoter usage.Mechanistically,we sought to explore the possible epigenetic regulatory role of RUNXOR with the help of experiments including chromosome conformation capture,DNA methylation analysis,RNA binding protein immunoprecipitation as well as chromatin immunoprecipitation.At last,the phenotypic changes of MCF-7 cell upon RUNXOR-RUNX1 activation were tested.Results:1.LncRNA RUNXOR correlated with RUNX1 mRNA transcribed from the distal promoter P1.To investigate the relation between RUNXOR and RUNX1,we compared the expression levels of RUNXOR and RUNX1 mRNA in a total of twelve cell lines of different tissue origins and found that there was a significant positive correlation between RUNXOR and RUNX1 expression levels(r=0.8790,p<0.0001).Besides,further analysis showed that RUNXOR was positively correlated with RUNX1 transcript from promoter Pl(r=-0.3774,p=0.1226).No correlation was found between RUNXOR and RUNX1 transcribed from promoter P2.2.LncRNA RUNXOR in situ activation preferentially upregulated RUNX1 transcription from the distal promoter P1.With the aid of the CRISPR-Cas9 editing system,we managed to insert a strong cytomegalovirus promoter(pCMV)in front of RUNXOR in MCF-7 cell lines and establish a cell model where lncRNA RUNXOR is endogenously activated in situ.RUNXI was significantly elevated both at the transcriptional and translational levels.Further dissection confirmed that the transcripts from P1 promoter were significantly increased upon RUNXOR in situ activation,while the transcripts derived from P2 promoter remained unchanged.At the protein level,the preferred activation of P1 promoter caused a shift to RUNXlc isoform dominant expression.3.LncRNA RUNXOR epigenetically regulated the alternative promoter usage of RUNX1 gene.Using a chromatin conformation capture(3C)approach,we found that RUNXOR IncRNA epigenetically activated the RUNX1 distal promoter P1 in cis by altering the local chromatin structure.Besides,the binding of IncRNA RUNXOR triggered DNA demethylation in the P1 CpG island and induced active histone modification markers around the P1 promoter.4.The activation of RUNXOR-RUNX1 axis in MCF-7 breast cancer cells significantly inhibited the migratory ability of cells and induced a trend to cell cycle arrest at G1/S.However,the cell proliferation rate and apoptosis were not significantly changed.Conclusions:1.LncRNA RUNXOR can epigenetically regulate the alternative promoter activation of its neighboring gene RUNX1.2.The activation of RUNXOR-RUNX1 axis in MCF-7 breast cancer cells significantly inhibited the migratory ability of cells and induced a trend to cell cycle arrest at G1/S,demonstrating its potential role as a tumor suppressor in this particular tumor subtype.3.The Cas9 mediated IncRNA in situ activation approach that we devised provided a better system for gain-of function research,and can be broadly used for both basic research and translational medicine.