Lysine-specific demethylase-2 is distinctively involved in brown and beige adipogenic differentiation.

Transcriptional and epigenetic regulation is fundamentally involved in initiating and maintaining progression of cellular differentiation. The 2 types of thermogenic adipocytes, brown and beige, are thought to be of different origins but share functionally similar phenotypes. Here, we report that lysine-specific demethylase 2 (LSD2) regulates the expression of genes associated with lineage identity during the differentiation of brown and beige adipogenic progenitors in mice. In HB2 mouse brown preadipocytes, short hairpin RNA-mediated knockdown (KD) of LSD2 impaired formation of lipid droplet-containing adipocytes and down-regulated brown adipogenesis-associated genes. Transcriptomic analysis revealed that myogenesis-associated genes were up-regulated in LSD2-KD cells under adipogenic induction. In addition, loss of LSD2 during later phases of differentiation had no obvious influence on adipogenic traits, suggesting that LSD2 functions during earlier phases of brown adipocyte differentiation. Using adipogenic cells from the brown adipose tissues of LSD2-knockout (KO) mice, we found reduced expression of brown adipogenesis genes, whereas myogenesis genes were not affected. In contrast, when LSD2-KO cells from inguinal white adipose tissues were subjected to beige induction, these cells showed a dramatic rise in myogenic gene expression. Collectively, these results suggest that LSD2 regulates distinct sets of genes during brown and beige adipocyte formation.-Takase, R., Hino, S., Nagaoka, K., Anan, K., Kohrogi, K., Araki, H., Hino, Y., Sakamoto, A., Nicholson, T. B., Chen, T., Nakao, M. Lysine-specific demethylase-2 is distinctively involved in brown and beige adipogenic differentiation.

In vitro evidence of NLRP3 inflammasome regulation by histone demethylase LSD2 in renal cancer: a pilot study.

NLRP3 pathway plays a vital role in the pathogenesis of different human cancers but still the regulation of NLRP3 pathway largely unknown. Therefore, we examined the levels of NLRP3 and its downstream components (caspase-1 and IL-1ß) and its relationship with histone modifiers in renal cancer pathogenesis. Total 30 cases of clear cell renal cell carcinoma (ccRCC), were studied for NLRP3, caspase-1 and IL-1ß expression using real-time PCR, which showed the augmented levels of all the three components of NLRP3 inflammasome pathway in ccRCC. Next, role of the FAD dependent monoamine oxidases (LSD2) and jumonji C (JmjC)-domain-containing, iron-dependent dioxygenases (KDM5A) histone demethylases were evaluated in regulation of NLRP3 inflammasome pathway in-vitro using RCC cell line. It was observed that silencing of KDM5A didn't alter the levels of neither of the NLRP3 component but inhibition of LSD2 showed significant effect on NLRP3 expression while no change in caspase-1 and IL-1ß levels. This study suggests that rather LSD2 not KDM5A lysine demethylase family might be involved in the regulation of NLRP3 inflammasome in cancer cells which could be useful for deciphering the future therapeutic targets for the disease.

Evaluation of phenylcyclopropylamine compounds by enzymatic assay of lysine-specific demethylase 2 in the presence of NPAC peptide.

Lysine-specific demethylase 2 (LSD2) demethylates mono- and dimethylated Lys-4 of histone H3 (H3K4me1 and H3K4me2). NPAC protein is known to interact with LSD2 and promote its H3K4 demethylase activity. In this study, we established a demethylation assay system that utilizes recombinant LSD2 in the presence of a synthetic NPAC peptide. Several phenylcyclopropylamine (PCPA)-based inhibitors were examined for their LSD2 inhibitory activity in the LSD2 enzymatic assay with the NPAC peptide. The assay results showed that the PCPA derivatives, including NCD41, selectively inhibited LSD1 in preference to LSD2.

KDM1 class flavin-dependent protein lysine demethylases.

Flavin-dependent, lysine-specific protein demethylases (KDM1s) are a subfamily of amine oxidases that catalyze the selective posttranslational oxidative demethylation of methyllysine side chains within protein and peptide substrates. KDM1s participate in the widespread epigenetic regulation of both normal and disease state transcriptional programs. Their activities are central to various cellular functions, such as hematopoietic and neuronal differentiation, cancer proliferation and metastasis, and viral lytic replication and establishment of latency. Interestingly, KDM1s function as catalytic subunits within complexes with coregulatory molecules that modulate enzymatic activity of the demethylases and coordinate their access to specific substrates at distinct sites within the cell and chromatin. Although several classes of KDM1-selective small molecule inhibitors have been recently developed, these pan-active site inhibition strategies lack the ability to selectively discriminate between KDM1 activity in specific, and occasionally opposing, functional contexts within these complexes. Here we review the discovery of this class of demethylases, their structures, chemical mechanisms, and specificity. Additionally, we review inhibition of this class of enzymes as well as emerging interactions with coregulatory molecules that regulate demethylase activity in highly specific functional contexts of biological and potential therapeutic importance.

Regulation of tissue factor pathway inhibitor-2 (TFPI-2) expression by lysine-specific demethylase 1 and 2 (LSD1 and LSD2).

Tissue factor pathway inhibitor-2 (TFPI-2) is a major inhibitor of extracellular matrix degradation. Decreases in TFPI-2 contribute to malignant tumor cell production, and TFPI-2 is a presumed tumor suppressor. TFPI-2 gene transcription is regulated by two epigenetic mechanisms: DNA methylation of the promoter and K4 methylation of histone 3 (H3). Lysine-specific demethylase 1 (LSD1) and LSD2 demethylate H3K4me2/1. LSD1 has been implicated in TFPI-2 regulation through both epigenetic mechanisms, but the involvement of LSD2 remains unknown. We prepared a monoclonal anti-LSD2 antibody that clearly distinguishes LSD2 from LSD1. Knockdown of LSD1 or LSD2 by siRNAs increased TFPI-2 protein and mRNA. Simultaneous knockdown of both LSD1 and LSD2 showed additive effects. Bisulfite sequencing revealed that CpG sites in the TFPI-2 promoter region were unmethylated. These results indicate that LSD2 also contributes to TFPI-2 regulation through histone modification, and that further studies of the involvement of LSD2 in tumor malignancy are warranted.

LSD2 Is an Epigenetic Player in Multiple Types of Cancer and Beyond.

Histone demethylases, enzymes responsible for removing methyl groups from histone proteins, have emerged as critical players in regulating gene expression and chromatin dynamics, thereby influencing various cellular processes. LSD2 and LSD1 have attracted considerable interest among these demethylases because of their associations with cancer. However, while LSD1 has received significant attention, LSD2 has not been recognized to the same extent. In this study, we conduct a comprehensive comparison between LSD2 and LSD1, with a focus on exploring LSD2's implications. While both share structural similarities, LSD2 possesses unique features as well. Functionally, LSD2 shows diverse roles, particularly in cancer, with tissue-dependent roles. Additionally, LSD2 extends beyond histone demethylation, impacting DNA methylation, cancer cell reprogramming, E3 ubiquitin ligase activity and DNA damage repair pathways. This study underscores the distinct roles of LSD2, providing insights into their contributions to cancer and other cellular processes.

The NPAC-LSD2 complex in nucleosome demethylation.

NPAC is a transcriptional co-activator widely associated with the H3K36me3 epigenetic marks present in the gene bodies. NPAC plays a fundamental role in RNA polymerase progression, and its depletion downregulates gene transcription. In this chapter, we review the current knowledge on the functional and structural features of this multi-domain protein. NPAC (also named GLYR1 or NP60) contains a PWWP motif, a chromatin binder and epigenetic reader that is proposed to weaken the DNA-histone contacts facilitating polymerase passage through the nucleosomes. The C-terminus of NPAC is a catalytically inactive dehydrogenase domain that forms a stable and rigid tetramer acting as an oligomerization module for the formation of co-transcriptional multimeric complexes. The PWWP and dehydrogenase domains are connected by a long, mostly disordered, linker that comprises putative sites for protein and DNA interactions. A short dodecapeptide sequence (residues 214-225) forms the binding site for LSD2, a flavin-dependent lysine-specific histone demethylase. This stretch of residues binds on the surface of LSD2 and facilitates the capture and processing of the H3 tail in the nucleosome context, thus promoting the H3K4me1/2 epigenetic mark removal. LSD2 is associated with other two chromatin modifiers, G9a and NSD3. The LSD2-G9a-NSD3 complex modifies the pattern of the post translational modifications deposited on histones, thus converting the relaxed chromatin into a transcriptionally refractory state after the RNA polymerase passage. NPAC is a scaffolding factor that organizes and coordinates the epigenetic activities required for optimal transcription elongation.

Histone Demethylase AMX-1 Regulates Fertility in a p53/CEP-1 Dependent Manner.

Histone methylation shapes the epigenetic configuration and adjusts multiple fundamental nuclear processes, including transcription, cell cycle control and DNA repair. The absence of histone demethylase LSD1/SPR-5 leads to progressive fertility defects as well as a reduction in brood size. Similarly, C. elegans LSD2 homolog AMX-1 has been implicated in regulating H3K4me2 and maintaining interstrand crosslinks (ICL) susceptibility. However, the mechanisms of how lack of AMX-1 induces sterility have not been addressed so far. This study investigated the histone demethylase AMX-1 in C. elegans and uncovered how amx-1 contributes to sterility in a p53/CEP-1 dependent manner. We show that while sterility in spr-5 mutants exhibited progressive over generations, amx-1 mutants displayed non-transgenerational fertility defects. Also, amx-1 mutants exhibited a reduced number of sperms and produced low brood size (LBS) or sterile worms that retain neither sperms nor germline nuclei, suggesting that fertility defects originated from germline development failure. Surprisingly, sterility exhibited in amx-1 was mediated by p53/CEP-1 function. Consistent with this result, upregulation of Piwi expression in amx-1 mutants suggested that AMX-1 is essential for germline development by regulating Piwi gene expressions. We propose that AMX-1 is required for proper Piwi expression and transposon silencing in a p53/CEP-1 dependent manner; thus, the absence of AMX-1 expression leads to defective meiotic development and sterility. This study elucidates how LSD2/AMX-1 contributes to sterility, therefore, expanding the boundaries of histone demethylase function.

The Role of LSD1 and LSD2 in Cancers of the Gastrointestinal System: An Update.

Epigenetic mechanisms are known to play a key role in cancer progression. Specifically, histone methylation involves reversible post-translational modification of histones that govern chromatin structure remodelling, genomic imprinting, gene expression, DNA damage repair, and meiotic crossover recombination, among other chromatin-based activities. Demethylases are enzymes that catalyse the demethylation of their substrate using a flavin adenine dinucleotide-dependent amine oxidation process. Lysine-specific demethylase 1 (LSD1) and its homolog, lysine-specific demethylase 2 (LSD2), are overexpressed in a variety of human cancer types and, thus, regulate tumour progression. In this review, we focus on the literature from the last 5 years concerning the role of LSD1 and LSD2 in the main gastrointestinal cancers (i.e., gastric cancer, liver cancer, pancreatic cancer, and colorectal cancer).

Lysine-specific histone demethylase 1B (LSD2/KDM1B) represses p53 expression to promote proliferation and inhibit apoptosis in colorectal cancer through LSD2-mediated H3K4me2 demethylation.

Epigenetic alterations have been reported to play critical roles in the development of colorectal cancer (CRC). However, the biological function of the lysine-specific histone demethylase 1B (LSD2/KDM1B) in CRC is not well understood. Therefore, we investigated the characteristics of LSD2 in CRC. We observed significant upregulation of LSD2 in CRC tissue compared to that in normal colorectal tissue. LSD2 promotes CRC cell proliferation and inhibits cell apoptosis through cell cycle regulation, promoting CRC progression both in vitro and in vivo. We found that LSD2 performs these functions by inhibiting the p53-p21-Rb pathway. Finally, we found that LSD2 directly binds to p53 and represses p53 expression via H3K4me2 demethylation at the p53 promoter. Our results revealed that LSD2 acts as an oncogene by binding and inhibiting p53 activity in CRC. Thus, LSD2 may be a new molecular target for CRC treatment.

Expression profile of H3K4 demethylases with their clinical and pathological correlation in patients with clear cell renal cell carcinoma.

OBJECTIVE: Clear cell renal cell carcinoma (ccRCC) is one of the deadly diseases with poor metastatic disease prognosis. There is an urgent need to explore the potential molecular markers which can improve the prognosis of the disease. Histone demethylases have emerged as a powerful tool for cancer prognosis and therapeutics during the last decade. The implications of demethylases of histone 3 lysine 4 (H3K4) in ccRCC are however unrevealed. We therefore evaluated the expression of H3K4 demethylases in ccRCC, with emphasis on their clinical significance as a prognostic marker. METHODS: Total 50 histopathological confirmed cases of ccRCC were enrolled in the study. The expression of seven H3K4 demethylases was determined by Real-Time PCR using gene specific primers and correlated with tumor stage, grade and metastasis. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the prognostic significance of H3K4 demethylases. RESULTS: The median age of the patients was 54 years with predominance of male patients by 2.6-fold. Among seven genes viz FBXL10, LSD1, LSD2, KDM5A, KDM5B, KDM5C and KDM5D analyzed, LSD2 was found to be significantly associated with tumor stage and metastasis. The optimal cut-off value for LSD2 was 3.2 as calculated from ROC curve analysis for metastasis as well as TNM stage with area under curve of 0.74 and 0.78 respectively. In addition, LSD2 expression showed significant positive correlation with LSD1 expression in tumor metastasis. CONCLUSION: The expression of LSD2 was associated with higher TNM stage and metastasis of the tumor and thus, might serve as a useful marker for ccRCC progression.

The Catalytic-Dependent and -Independent Roles of Lsd1 and Lsd2 Lysine Demethylases in Heterochromatin Formation in Schizosaccharomyces pombe.

In eukaryotes, heterochromatin plays a critical role in organismal development and cell fate acquisition, through regulating gene expression. The evolutionarily conserved lysine-specific demethylases, Lsd1 and Lsd2, remove mono- and dimethylation on histone H3, serving complex roles in gene expression. In the fission yeast Schizosaccharomyces pombe, null mutations of Lsd1 and Lsd2 result in either severe growth defects or inviability, while catalytic inactivation causes minimal defects, indicating that Lsd1 and Lsd2 have essential functions beyond their known demethylase activity. Here, we show that catalytic mutants of Lsd1 or Lsd2 partially assemble functional heterochromatin at centromeres in RNAi-deficient cells, while the C-terminal truncated alleles of Lsd1 or Lsd2 exacerbate heterochromatin formation at all major heterochromatic regions, suggesting that Lsd1 and Lsd2 repress heterochromatic transcripts through mechanisms both dependent on and independent of their catalytic activities. Lsd1 and Lsd2 are also involved in the establishment and maintenance of heterochromatin. At constitutive heterochromatic regions, Lsd1 and Lsd2 regulate one another and cooperate with other histone modifiers, including the class II HDAC Clr3 and the Sirtuin family protein Sir2 for gene silencing, but not with the class I HDAC Clr6. Our findings explore the roles of lysine-specific demethylases in epigenetic gene silencing at heterochromatic regions.

Investigating the role of LSD2 as an epigenetic regulator in Ewing sarcoma.

Ewing sarcoma is the second most common solid bone malignancy diagnosed in pediatric and young adolescent populations. Despite aggressive multi-modal treatment strategies, 5-year event-free survival remains at 75% for patients with localized disease and 20% for patients with metastases. Thus, the need for novel therapeutic options is imperative. Recent studies have focused on epigenetic misregulation in Ewing sarcoma development and potential new oncotargets for treatment. This project focused on the study of LSD2, a flavin-dependent histone demethylase found to be overexpressed in numerous cancers. We previously demonstrated that Ewing sarcoma cell lines are extremely susceptible to small molecule LSD1 blockade with SP-2509. Drug sensitivity correlated with the degree of LSD2 induction following treatment. As such, the purpose of this study was to determine the role of LSD2 in the epigenetic regulation of Ewing sarcoma, characterize genes regulated by LSD2, and examine the impact of SP-2509 drug treatment on LSD2 gene regulation. Genetic depletion (shRNA) of LSD2 significantly impaired oncogenic transformation with only a modest impact on proliferation. Transcriptional analysis of Ewing sarcoma cells following LSD2knockdown revealed modulation of genes primarily involved in metabolic regulation and nervous system development. Gene set enrichment analysis showed that SP-2509 does not impact LSD2 targeted genes. Although there are currently no small molecule agents that specifically target LSD2, our results support further investigations into agents that can inhibit this histone demethylase as a possible treatment for Ewing sarcoma.

Molecular signature of the imprintosome complex at the mating-type locus in fission yeast.

Genetic and molecular studies have indicated that an epigenetic imprint at mat1, the sexual locus of fission yeast, initiates mating type switching. The polar DNA replication of mat1 generates an imprint on the Watson strand. The process by which the imprint is formed and maintained through the cell cycle remains unclear. To understand better the mechanism of imprint formation and stability, we characterized the recruitment of early players of mating type switching at the mat1 region. We found that the switch activating protein 1 (Sap1) is preferentially recruited inside the mat1M allele on a sequence (SS13) that enhances the imprint. The lysine specific demethylases, Lsd1/2, that control the replication fork pause at MPS1 and the formation of the imprint are specifically drafted inside of mat1, regardless of the allele. The CENP-B homolog, Abp1, is highly enriched next to mat1 but it is not required in the process. Additionally, we established the computational signature of the imprint. Using this signature, we show that both sides of the imprinted molecule are bound by Lsd1/2 and Sap1, suggesting a nucleoprotein protective structure defined as imprintosome.

Structural insight into inhibitors of flavin adenine dinucleotide-dependent lysine demethylases.

Until 2004, many researchers believed that protein methylation in eukaryotic cells was an irreversible reaction. However, the discovery of lysine-specific demethylase 1 in 2004 drastically changed this view and the concept of chromatin regulation. Since then, the enzymes responsible for lysine demethylation and their cellular substrates, biological significance, and selective regulation have become major research topics in epigenetics and chromatin biology. Many cell-permeable inhibitors for lysine demethylases have been developed, including both target-specific and nonspecific inhibitors. Structural understanding of how these inhibitors bind to lysine demethylases is crucial both for validation of the inhibitors as chemical probes and for the rational design of more potent, target-specific inhibitors. This review focuses on published small-molecule inhibitors targeted at the two flavin adenine dinucleotide-dependent lysine demethylases, lysine-specific demethylases 1 and 2, and how the inhibitors interact with the tertiary structures of the enzymes.

Clues on the function of Manduca sexta perilipin 2 inferred from developmental and nutrition-dependent changes in its expression.

Cellular triglycerides (TG) are stored in cytosolic lipid droplets (LDs). Perilipins (PLIN) are a group of LD-proteins that play important roles in the assembly and transport of LDs and in TG metabolism. Two members of the PLIN family are found in insects (PLIN1 & 2 or Lsd1 & 2). We have cloned and expressed Manduca sexta PLIN2 (MsPLIN2), and studied developmental and nutritional changes in the expression of PLIN2. Nutritional changes induced fast alterations in PLIN2 mRNA and protein levels in fat body and midgut of the feeding larvae. The relationship observed between PLIN2 expression and TG synthesis in both larval fat body and midgut suggests that PLIN2 is needed when tissues are accumulating TG. However, when the fat body was storing TG at maximal capacity, MsPLIN2 levels declined. This unexpected finding suggests the occurrence of alternative mechanism/s to shield TG from the action of lipases in M. sexta LDs. In addition, it implies that the cellular level of lipid storage could be modulating MsPLIN2 expression and/or degradation. The study also confirmed that MsPLIN2 was most abundant in the adult fat body, which is characterized by a high rate of TG hydrolysis and lipid mobilization. Whether MsPLIN2 is directly involved in lipolysis and/or the secretion of lipids in the fat body of adult of M. sexta is unknown at this time. Nonetheless, the coexistence of high PLIN2 and lipolysis levels suggests a complex role for MsPLIN2. Altogether, we found that MsPLIN2 is needed when the synthesis of glycerides, DG and TG, is active even if the insect is accumulating or consuming TG.

Functional characterization of lysine-specific demethylase 2 (LSD2/KDM1B) in breast cancer progression.

Flavin-dependent histone demethylases govern histone H3K4 methylation and act as important chromatin modulators that are extensively involved in regulation of DNA replication, gene transcription, DNA repair, and heterochromatin gene silencing. While the activities of lysine-specific demethylase 1 (LSD1/KDM1A) in facilitating breast cancer progression have been well characterized, the roles of its homolog LSD2 (KDM1B) in breast oncogenesis are relatively less understood. In this study, we showed that LSD2 protein level was significantly elevated in malignant breast cell lines compared with normal breast epithelial cell line. TCGA- Oncomine database showed that LSD2 expression is significantly higher in basal-like breast tumors compared to other breast cancer subtypes or normal breast tissue. Overexpression of LSD2 in MDA-MB-231 cells significantly altered the expression of key important epigenetic modifiers such as LSD1, HDAC1/2, and DNMT3B; promoted cellular proliferation; and augmented colony formation in soft agar; while attenuating motility and invasion. Conversely, siRNA-mediated depletion of endogenous LSD2 hindered growth of multiple breast cancer cell lines while shRNA-mediated LSD2 depletion augmented motility and invasion. Moreover, LSD2 overexpression in MDA-MB-231 cells facilitated mammosphere formation, enriched the subpopulation of CD49f+/EpCAM- and ALDHhigh, and induced the expression of pluripotent stem cell markers, NANOG and SOX2. In xenograft studies using immune-compromised mice, LSD2-overexpressing MDA-MB-231 cells displayed accelerated tumor growth but significantly fewer lung metastases than controls. Taken together, our findings provide novel insights into the critical and multifaceted roles of LSD2 in the regulation of breast cancer progression and cancer stem cell enrichment.