LSD1 and the chemistry of histone demethylation
Introduction
One of the surprises in determining the sequence of the human genome was how relatively few genes (ca. 25 000) are encoded by our DNA [1]. As a result, biologists have come to appreciate more than ever that the informational diversity that leads to our complexity stems from post-transcriptional mechanisms including RNA splicing and protein post-translational modifications. The latter allows for a rich variety of covalent protein structural changes that includes phosphorylation, acetylation, ubiquitylation, sulfation, glycosylation, lipidation, and methylation [2]. These changes in protein chemical structure often result in profound influences on the mechanism and function of proteins in cells.
Core histone proteins are highly conserved and play a crucial role in modulating chromatin structure and DNA accessibility for replication, repair, and transcription [3]. There has been intense interest in mapping and characterizing the histone post-translational modifications that guide chromatin remodeling and account for epigenetics. In this regard, the protein lysine modifications have a special place in the history of gene regulation. Allfrey et al. demonstrated in the 1960s that histone lysine acetylation was often found in chromatin that was in an activated state [4]. Histone lysine methylation was proposed to be reciprocal with acetylation in structure and function. We now know that the effects of lysine acetylation and methylation are more variegated than these early concepts and depend on specific modification sites as well as additional factors at specific genes. The progress of research in this field has been catapulted by a series of discoveries of key enzymes that catalyze histone lysine acetylation and deacetylation [5, 6] on the one hand and methylation and demethylation [7, 8••] on the other.
The first specific histone lysine methyltransferases were reported in 2000 and as expected were S-adenosyl methionine dependent enzymes [7]. There are at least two classes of histone lysine methyltransferases, the SET domain containing protein family [9] and the non-SET domain protein analogs of DOT1 [10, 11, 12]. Several of these methyltransferases are quite specific for their targeted lysine and preferentially add one, two, or three methyl groups. For many years, it was uncertain if methylation marks on histones were ‘permanent’. Unlike the reversibility of most post-translational modifications, which involve hydrolytic reactions, it seemed less likely that lysine methylation would be cleaved in this manner because of the kinetic stability of the N–Me bond. In 1973 Paik and Kim demonstrated the enzymatic demethylation of N-methylated calf thymus histones, though they were unable to ultimately identify the corresponding enzyme [13].
Section snippets
Lysine demethylases
In late 2004, it was reported that a flavin-dependent amine oxidase that appeared to be a polyamine oxidase homolog, LSD1, was capable of selective demethylation of Lys-4 of histone H3 [8••]. This study laid to rest the debate as to whether or not lysine methylation was a static epigenetic mark and opened up a new chapter in the chromatin field. Since the initial description of LSD1, there has been a frenzied pace of molecular biology discovery in reversible protein methylation. At least 10
LSD1 reaction mechanism
LSD1 is a member of the monoamine oxidase family based on sequence and recent X-ray crystal structures [8••, 28], and there has been a great deal of research into the detailed catalytic mechanisms of these enzymes. The FAD cofactor is oxidized by molecular oxygen, presumably by a single electron mechanism. Studies with LSD1 suggest that this flavin oxidation is rapid [29] on the basis of changes in the flavin optical spectrum that show classic FAD absorbance maxima at approximately 460 and 380
LSD1 substrate specificity
LSD1 is most closely related to the polyamine oxidase superfamily and LSD1 was originally tested with various small molecule polyamines as potential substrates, but these compounds showed minimal or undetectable turnover under the reaction conditions [8••]. Various methyl-lysine peptides derived from histones were then investigated as possible substrates, including mono-methylated, di-methylated, and tri-methylated Lys-4 H3 tail peptides, full-length Lys-4-methylated histone H3, and
Structural studies on LSD1 in complex with a histone analog suicide inhibitor
Insertion of various functional groups including cyclopropyls and propargyls into monoamine oxidase substrates converts these compounds into mechanism-based inactivators [38, 39]. Substitutions at the epsilon position of Lys-4 of histone H3 peptides including propargylamine, aziridine, and cyclopropylamine have been synthesized and the propargylamine-containing peptide behaves as a classical mechanism-based inactivator [40•]. On the basis of a combination of mass spectrometry and optical and
In vivo and nucleosomal targeting
Relatively little is known about the structural basis of demethylation of chromatin by LSD1 in vivo. However, it appears that CoREST plays an important role in targeting histone H3 demethylation in the context of nucleosomes [34, 35]. Knockdown of CoREST greatly reduces the efficiency of LSD1-mediated histone demethylation in vivo, and biochemical studies with nucleosome substrates have confirmed this behavior. On the basis of NMR and mutagenesis experiments it has been proposed that CoREST may
LSD1 small molecule inhibitors
LSD1 as a therapeutic target for small molecule inhibitors is plausible in analogy to the now clinically validated epigenetic HDAC enzyme class. Along these lines, the cyclopropylamine-containing monoamine oxidase inhibitor tranylcypromine was proposed to inhibit LSD1 [44•] and further characterized as a mechanism-based inactivator of LSD1 [45, 46]. It is noteworthy that a cyclopropylamine substrate analog of histone H3 failed to induce inactivation of LSD1, suggesting tranylcypromine adopts a
Conclusion and future challenges
It is difficult to overstate the briskness of research in the histone demethylation field set off by the initial report of LSD1 less than 3 years ago [8••]. The flavin-dependent and iron-dependent demethylases appear to play key roles in gene regulation and cellular growth and differentiation. Still, there are many remaining challenges. How precisely do these demethylases catalyze reactions and recognize their substrates, and how does the in vivo context modify and regulate these interactions?
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
We are grateful to our colleagues and collaborators for helpful advice and discussions including Yang Shi, Hongtao Yu, Maojun Yang, Xin Liu, Ronen Marmorstein, Larry Szewczuk, Rong Huang, and Marc Holbert. We thank the NIH for financial support.
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