About the lab

1. The deciphering of the role of RNA epigenetics, including RNA editing and RNA methylation in the regulation of gene expression and cell fate.

A major area of interest is RNA editing and modifications.

An extensive repertoire of modifications is known to underlie the versatile coding, structural and catalytic functions of RNA, with more than one hundred modifications characterized so far using biochemical approaches. We were the first to demonstrate the global nature of Adenosine to Inosine (A-to-I) RNA  editing (Levanon et al. Nature Biotechnology 2004). We are studying the role of RNA editing in basic cellular regulatory activities and the relation of abnormal editing with a variety of disease states. We found that alterations in RNA editing are frequent in various types of cancer and we are studying the pathogenic mechanisms by which abnormal editing of messenger RNA and microRNAs contribute to the malignant phenotype (Paz et al Genome Research 2009, Dominissini et al Carcinogenesis 2012, Dominissini et al Journal of Clinical Investigation 2012). We demonstrated that RNA editing is significantly more extensive in humans compared to other organisms and in particular that editing in humans is higher than that of non-human primates and suggested a role for editing in higher brain functions (Eisenberg et al Nucleic Acid Research 2005, Paz-Yaacov et al PNAS 2009). Currently we are studying mouse and zebrafish models where the editing machinery is disrupted to shed light on the role of this process in normal physiology and its relevance to brain, liver and immune system diseases.
Our group pioneered the study of widespread methyl-6-Adenosine (m6A) modification in mRNA. N6-methyladenosine (m6A) is the most common internal mRNA modification found in eukaryotic organisms as well as in RNA of nuclear-replicating viruses. Since m6A was first described in mRNA almost four decades ago, only a few sites have been mapped in cellular and viral RNA. Although biochemical studies indicate that N6-methyl adenosine (m6A) is the most prevalent internal modification in messenger RNA, an in-depth study of its distribution and functions has been impeded by a lack of robust analytical methods. We developed a novel approach, m6A-seq, based on antibody-mediated capture and massively parallel sequencing (Dominissini et al Nature 2012, Dominissini et al Nature Protocols 2013, Fu et al Nat Rev Genet 2014). Using this methodology we characterized the human and mouse methylomes and identified over 20,000 m6A sites characterized by a typical consensus in the transcripts of more than 7,000 human genes. Sites preferentially appear in two distinct landmarks—around stop codons and within long internal exons—and are highly conserved between human and mouse. Although most sites are well preserved across normal and cancer tissues and in response to various stimuli, a subset of  stimulus-dependent, dynamically modulated sites was identified. Silencing the m6A methyl transferase significantly affected gene expression and alternative splicing patterns. These findings therefore point to RNA decoration by m6A as a fundamental player in regulation of gene expression. Furthermore, our work not only identified and mapped the RNA methylated sites but also identified a new group of m6A binding proteins that are involved in "reading" the modified RNA sites and execute their biological effects. We recently identified, using cells and mice where the main methyltransferase METTL3 was depleted, a critical role for m6A RNA modification in early embryonic development, specifically in the transition of naïve embryonic stem cells to primed epiblast cells.
 

2. The study of transposable genetic elements in cancer

     and neurodevelopmental disorders
Our group was a pioneer in the study of repetitive DNA elements that at the time were known as "junk DNA". We showed that these elements transpose in the human genome and result in activation of cellular oncogenes. In a major article in Nature, (Rechavi et al Nature 1982) we showed that the c-mos oncogene is activated by the insertion of a repetitive element. A second (Kuff et al Nature1983) paper identified the inserted element as an intracisternal A particle element and characterized the mechanism of oncogene activation (Canaani et al PNAS 1983). A third Nature (Cohen et al 1983) article studied the rearrangement of c-mos in Multiple Myeloma and studied the cooperation of this oncogene with the c-myc oncogene.
Another example of oncogene activation was found in the transmissible venereal tumor (TVT) where a LINE element was inserted upstream of the c-myc oncogene and led to its activation (Katzir et al PNAS 1985, Amariglio et al PNAS 1991). A fourth Nature publication discussed the relevance of oncogene activation by transposable elements in cancer, in particular breast cancer (Rechavi et al Nature 1988).
The years that followed confirmed that indeed transposition of mobile genetic elements contributes significantly to genomic evolution. The genomic instability that characterizes cancer is associated with enhanced genomic rearrangements resulting in oncogene activation and tumor suppressor silencing mediated by repetitive elements. The human genome project showed that repetitive elements comprise about half of the genome while protein coding genes comprise only 2% of the genome. The study of repetitive elements in cancer which we pioneered is now becoming very central in the post genomic era.

Neural progenitor cells undergo somatic retrotransposition events, mainly involving L1 elements, which can be potentially deleterious. We recently analyzed the whole genomes of 20 brain samples and 80 non-brain samples, and characterized the retrotransposition landscape of patients affected by a variety of neurodevelopmental disorders including Rett syndrome, tuberous sclerosis, ataxia-telangiectasia and autism (Cell Research 2018). The number of retrotranspositions in brain tissues was found to be higher than that observed in non-brain samples and even higher in pathologic vs normal brains. The majority of somatic brain retrotransposons integrate into pre-existing repetitive elements, preferentially A/T rich L1 sequences, resulting in nested insertions. Our findings document the fingerprints of encoded endonuclease independent mechanisms in the majority of L1 brain insertion events. The insertions are “non-classical” in that they are truncated at both ends, integrate in the same orientation as the host element, and their target sequences are enriched with a CCATT motif in contrast to the classical endonuclease motif of most other retrotranspositions. We showed that L1Hs elements integrate preferentially into genes associated with neural functions and diseases. We propose that pre-existing retrotransposons act as “lightning rods” for novel insertions, which may give fine modulation of gene expression while safeguarding from deleterious events. Overwhelmingly uncontrolled retrotransposition may breach this safeguard mechanism and increase the risk of harmful mutagenesis in neurodevelopmental disorders.


3. Genetically non-identical tumors


The study of the canine transmissible venereal tumor (TVT), where the c-myc oncogene was shown to be activated by a transposable element led us to another unexpected finding. TVT was known since the 19th century to be sexually transmitted among canines and was attributed to an infectious agent. Demonstration that  the rearranged c-myc oncogene in tumors from different continents is identical led us to the amazing conclusion that all TVT tumors originate  from a single malignant cell that was transmitted between dogs beyond major histocompatibility barriers due to lack of expression of beta2 microglobulin. The unraveling of this unusual experiment of nature (Amariglio et al PNAS 1991) led us to propose that cancer can be transmitted from one individual to another as a cell mediated "infectious disease". This challenging hypothesis was supported years later (Murgia et al Cell 2006; Murchison et al Science 2014) using the same molecular approach we described and by the study of another naturally occurring infectious cancer that was shown to disseminate among the Tasmanian devil and actually led to their extinction. These studies were shown to be relevant to many types of

cancer in humans. These include donor type tumors following bone marrow and solid organ transplants, maternal to fetal transmission of tumors, twin to twin transmission of leukemia and tumor development following accidental injection of cancer cells. Recently we described and in depth molecularly characterized the first example of a human genetically non-identical brain tumor developing following therapeutic transplantation of fetal neural stem cells. This work (Amariglio et al PLoS Medicine 2009), and a second case we described in Nature Medicine (Amariglio et al 2010) demonstrated the need for very extensive research to make stem cell therapies beneficial while avoiding the risk of tumor induction. Moreover this work contributed to the concept of cancer stem cells and increased our understanding of brain tumor development.

 

4. The p53 tumor suppressor gene

Our group made a significant contribution to the understanding of the role of p53 in normal hematopoiesis and lymphopoiesis and in particular to the role of p53 in leukemias and lymphomas. We identified a regulatory loop by which p53 regulates the expression of the transcriptional repressor BCL6 which is essential for the precise regulation of the somatic hypermutation machinery while preventing oncogene activation by excessive mutagenesis. This regulatory loop was shown to be deregulated in the majority of diffuse large B cell lymphomas, the most common lymphomas (Margalit et al Blood 2006).
p53 was shown to be induced by hypoxia and a lot of supporting information suggests that p53 mutations are essential for tumor cell survival under hypoxic conditions. An example of the creative and innovative thinking of Rechavi is the elegant study of the Spalax p53. This underground living mole is the mammal that can resist extreme hypoxia conditions best. We showed that the Spalax p53 is characterized by evolution-selected mutations that are identical to those frequently acquired during solid tumor evolution. The native Spalax p53, found to be similar to many mutant human p53 genes present in tumors, was shown to be defective in its ability to induce apoptosis (Ashur-Fabian et al PNAS 2004).
High throughput gene expression analysis using DNA microarrays in our laboratory identified the direct and indirect targets of p53, which are involved in apoptosis and cell cycle arrest and elucidated the molecular pathways, relevant to normal cell response to stress, that are perturbed in cancer (Kannan et al Oncogene 2001).

 

 5. Genetic and genomic studies relevant to cancer and genetic diseases

Our group operates a center for advanced medical genomics equipped with next generation sequencers (Illumina HiSeq 2500, MiSeq and Ion Proton) as well as a microarray platform (Affymetrix) and an in house bioinformatics unit. The genomic center is involved in the unraveling of the molecular basis of a variety of human disorders ranging from various types of cancer to neurodegenerative disorders. We were involved in the identification of new causative genes and mutations of several new genetic syndromes and in the study of the biochemical and functional consequences of these aberrations. The new mutated genes identified include:

 

  1. STIM1- associated with a syndrome of immunodeficiency and autoimmunity (Picard C et al. NEJM 2009)

  2. SOBP - syndromic and non syndromic intellectual disability (Birk E et al.   Am J Hum Genet 2010)

  3. PIGN - multiple congenital anomalies -hypotonia-seizures syndrome (Maydan G et al. J. Med Genet. 2011)

  4. GPD1- a syndrome of transient infantile hypertriglyceridemia, fatty liver, and hepatic fibrosis (Basel-Vanagaite L  et al. Am J Hum Genet. 2012)

  5. CC2D1A - in autosomal recessive non syndromic mental retardation (Basel-Vanagaite L J et al.  Med Genet. 2006)

  6. nup62 - in autosomal recessive infantile bilateral striatal necrosis (Basel-Vanagaite L et al. Ann Neurol. 2006)

  7. EFTs  - in encephalomyopathy and hypertrophic cardiomyopathy (Smeitink JA  et al. Am J Hum Genet. 2006)

  8. VP45 -in familial neutropenia (Vilboux T et al.  NEJM 2013)

  9. Mutations in STN1 cause Coats plus syndrome and are associated with genomic and telomere defects (Simon AJ et al. J Exp Med 2016)