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Reversible RNA and DNA Methylation

Research Summary

Chuan He's group discovered the first RNA demethylase that reverses the most prevalent internal messenger RNA methylation in mammalian cells. He is working toward revealing biological functions and understanding pathways and mechanisms associated with reversible RNA methylation in various organisms. He also develops and applies enabling technologies to understand active DNA demethylation in modulating gene expression.

The mammalian genome is not merely a static combination of genetic information. Reversible chemical modifications of DNA and histones, the epigenetic modifications, contribute significantly to dynamic regulation of gene expression. Research in my group centers on understanding dynamic modifications of RNA and DNA, in particular, the roles of demethylation of RNA and DNA in biological regulation.

Reversible RNA Methylation
Genetic information flows from DNA to RNA and then to protein in the central dogma. Reversible epigenetic modifications (e.g., methylation) of DNA and histones are well known to play key roles in the regulation of gene expression. Prior to our work, however, there were no established examples of reversible chemical modifications of RNA that affect gene expression. Cellular RNAs contain more than 100 structurally distinct post-transcriptional modifications at thousands of sites, serving versatile coding, structural, and catalytic functions. We hypothesize that some of these RNA modifications can be dynamic/reversible and may have regulatory roles analogous to reversible DNA and protein modifications. Recently we have discovered two functionally significant RNA demethylases that oxidatively demethylate N6-methyladenosine (m6A), the most prevalent internal modification in human messenger RNA (mRNA). We found that FTO, a protein associated with human fat mass and obesity, and its homolog, ALKBH5, a protein critical for mouse fertility, catalyze the demethylation of m6A in human mRNA. The knockdown of FTO and deficiency of ALKBH5 lead to increased m6A in polyadenylated RNA in various human cell lines and tissues from the Alkbh5 -/- mice, respectively, confirming RNA demethylation in vivo. The significant phenotypes associated with these two RNA demethylases, the first discovered so far, strongly indicate that RNA demethylation plays critical roles in biological regulation.

Figure: Reversible methylation of RNA and DNA in gene expression regulation.

To fully understand the roles through which reversible RNA methylation affects protein expression, we are identifying and characterizing selective m6A-binding proteins. Our recent results indicate that several such proteins specifically recognize m6A in mRNA and/or noncoding RNA and affect RNA stability, cellular localization, and nuclear export as well as translation. Only a portion of nuclear RNAs with a consensus sequence are methylated by a putative RNA methyltransferase complex. We are also characterizing the whole mammalian methyltransferase complex to understand its selectivity, the connection to transcription, and the consequences of nuclear RNA m6A methylation. We hypothesize that reversible RNA methylation, analogous to reversible DNA and histone modifications, affects protein expression and provides a rapid-response pathway to various stimuli and signals. Research in my lab aims to elucidate the exact function, pathway, and mechanism of dynamic m6A modification of RNA, which could influence broad biological processes.

5-Methylcytosine Oxidation and Active DNA Demethylation
Methylation of cytosine to form 5-methylcytosine (5mC) on genomic DNA plays important roles in controlling gene expression. Although DNA methylation has been well studied, the reverse process has only recently been revealed in mammalian cells. A group of iron(II)/αKG-dependent dioxygenases, the TET proteins, use dioxygen to oxidize 5mC to 5-hydroxymethylcytosine (5hmC) and then to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). The resulting 5fC and 5caC can be recognized by mammalian DNA glycosylase, thymine DNA glycosylase (TDG),  in a base excision repair process to convert back to cytosine, providing an active demethylation pathway. My laboratory has spearheaded the development of new technologies to label these modified cytosines in order to determine their exact genomic location and study the active demethylation process. The new technologies include: (1) a selective chemical labeling strategy that uses bacterial phage β-glucosyltransferase (βGT) to place a biotin or any chemical group on 5hmC to profile and detect it in genomic DNA; (2) a TET-assisted bisulfite-sequencing (TAB-Seq) method that is capable of single-base-resolution sequencing of 5hmC as well as determining the relative abundance of both 5hmC and 5mC when combined with traditional bisulfite sequencing; (3) a strategy of 5fC reduction for its genome-wide profiling; and (4) chemical modification–assisted bisulfite sequencing (CAB-Seq) methods to obtain single-base-resolution information about 5fC and 5caC in genomic DNA. Many of these methods have been commercialized, providing the community with tools to investigate 5mC oxidation and active DNA demethylation in various systems.

Applying our new methods, we have discovered that active DNA demethylation occurs genome-wide in mammalian systems, in particular at poised and active enhancer sites, many of which undergo dynamic methylation and demethylation. The shift of this dynamic equilibrium at these distal regulatory elements appears to largely contribute to changes in gene expression upon cell differentiation or development. We plan to explore the scope and significance of active DNA demethylation in specific biological transformations or human diseases. Besides serving as intermediates in active DNA demethylation, 5hmC, 5fC, and even 5caC could themselves serve as potential epigenetic marks that interact with potential binder proteins to directly exert regulatory functions. We are particularly interested in investigating potential functional roles of these newly discovered cytosine derivatives in various mammalian systems.

Selective Metal Recognition and Metal Trafficking
All cells must take up, transport, and store transition metal ions as essential cofactors for various biological processes. While these transition metal ions are essential for cell survival, their concentrations in biological fluids must be monitored and regulated constantly to avoid toxic side effects. My laboratory studies the fundamental mechanisms of selective metal recognition and regulation in biological systems. Building on our understanding of metal-trafficking pathways in mammalian cells, we also develop new therapeutic strategies that target metal trafficking with the hope of curing human diseases such as cancer.

Grants from the National Institutes of Health and National Science Foundation provided partial support for these projects.

As of February 22, 2016

Scientist Profile

Investigator
The University of Chicago
Chemical Biology, Genetics