br Expression and Purification of LSD Proteins br In
Expression and Purification of LSD1 Proteins
In Vitro Enzymatic Assays
Analysis of LSD1 Demethylase Activity
Conflict of Interest Statement: Y.S. is a cofounder of Constellation Pharmaceuticals, Inc. and is a member of its scientific advisory board. Y.S. is also a consultant for Active Motif, Inc.
Although histone methylation was originally thought to be an irreversible Ready-to-use Cell Proliferation Reagent, WST-1 modification, biochemical characterization of KDM1A/LSD1, a flavin-dependent amine oxidase, led to the discovery of the first known histone demethylase. KDM1A is a component of the CoREST transcriptional repressor complex and exhibits H3K4me2/me1 demethylase activity , . Subsequent studies showed that KDM1A also associates with nuclear receptors, where it functions as a coactivator through demethylation of repressive H3K9me2 , . The KDM1A homolog KDM1B/LSD2 is also an H3K4me2 demethylase , . However, KDM1A/B cannot remove trimethyl groups from modified lysines, which suggests that alternative enzymatic activities exist. Indeed, several research groups have discovered that the Jumonji C (JmjC) domain family of Fe(II)- and α-ketoglutarate–dependent dioxygenases function as histone demethylases in a mechanism that involves oxidative hydroxylation by radical attack of the methyl group (for review, Kampranis and Tsichlis, 2009 and Kooistra and Helin, 2012 ). The human genome encodes approximately 30 JmjC domain histone demethylases that, based on structural homology of the JmjC domain, can be further divided into groups with activities toward H3K4, H3K9, H3K27, and H3K36 modifications. Several of these demethylases have also been shown to act on nonhistone substrates , . Considering the fast pace of advancements in the field of epigenetics, it is likely that additional enzymatic systems capable of demethylating histones will be described in the future. Mass spectrometry–based studies revealed that lysine-specific histone demethylases participate in multiprotein complexes to regulate gene expression, heterochromatin formation, and genome organization , . In recent years, several of these enzymes have been implicated in the epigenetic regulation of normal hematopoiesis, whereas their deregulation has been linked to the development of leukemias. Given the reversibility of histone methylation, understanding the context-dependent roles of histone demethylases will be critical for the development of novel therapies. Histone demethylases in hematopoiesis In murine embryogenesis, definitive hematopoiesis commences around embryonic day 10.5 from the hemogenic endothelium (HE) of the aorta–gonad–mesonephros (AGM) region. The hematopoietic stem and progenitor cells (HSPCs) that arise from the AGM are the first to harbor multilineage reconstitution capacity 9, 10. These HSPCs subsequently migrate to the fetal liver, where they expand before homing to the bone marrow, the permanent site for hematopoiesis throughout adulthood 9, 10. In adult mice, HSPCs reside in a compartment defined by a lack of lineage-specific markers (Linneg) and positive for both c-Kit and Sca-1 (LinnegKS+), whereas long-term hematopoietic stem cells (HSCs) are further identified by the presence of CD150 and the absence of CD48 and can rescue myeloablated mice from hematopoietic failure and establish long-term multilineage reconstitution . The first insight into the role of histone demethylation in hematopoietic development began with the finding that deletion of Kdm1a perturbs terminal differentiation of erythroid, megakaryocytic, and granulocytic cells by derepressing Gfi-1/1b-lineage-specific transcriptional programs . Further studies revealed an indispensable role for Kdm1a in the emergence of HSCs from the AGM through silencing the endothelial program within the HE in mice and zebrafish 13, 14, suggesting an evolutionarily conserved role in definitive hematopoiesis. Conditional deletion of Kdm1a in fetal (Vav1) and adult (Mx1) HSPCs resulted in severe pancytopenia and compromised terminal differentiation of granulocytic and erythroid lineages 15, 16. At the molecular level, Kdm1a binds enhancers of genes that regulate self-renewal and lineage commitment pathways and demethylates H3K4me1/2 as part of the CoREST repressive complex 12, 13, 15.