LSD1 Target Genes Research Articles

Tranylcypromine-Based LSD1 Inhibitors: Structure-Activity Relationships, Antiproliferative Effects in Leukemia, and Gene Target Modulation.

LSD1 is a lysine demethylase highly involved in initiation and development of cancer. To design highly effective covalent inhibitors, a strategy is to fill its large catalytic cleft by designing tranylcypromine (TCP) analogs decorated with long, hindered substituents. We prepared three series of TCP analogs, carrying aroyl- and arylacetylamino (1 a-h), Z-amino acylamino (2 a-o), or double-substituted benzamide (3 a-n) residues at the C4 or C3 position of the phenyl ring. Further fragments obtained by chemical manipulation applied on the TCP scaffold (compounds 4 a-i) were also prepared. When tested against LSD1, most of 1 and 3 exhibited IC50 values in the low nanomolar range, with 1 e and 3 a,d,f,g being also the most selective respect to monoamine oxidases. In MV4-11 AML and NB4 APL cells compounds 3 were the most potent, displaying up to sub-micromolar cell growth inhibition against both cell lines (3 a) or against NB4 cells (3 c). The most potent compounds in cellular assays were also able to induce the expression of LSD1 target genes, such as GFI-1b, ITGAM, and KCTD12, as functional read-out for LSD1 inhibition. Mouse and human intrinsic clearance data highlighted the high metabolic stability of compounds 3 a, 3 d and 3 g. Further studies will be performed on the new compounds 3 a and 3 c to assess their anticancer potential in different cancer contexts.

Abstract A169: Combination of TPC-144, a reversible LSD1 inhibitor, and a hypomethylating agent resulted in synergistic antitumor efficacy in preclinical models of AML

Abstract Background: Lysine-specific demethylase 1A (LSD1/KDM1A) is a flavin adenine dinucleotide (FAD)-dependent histone demethylase that specifically modifies histone H3 lysine 4 and lysine 9. LSD1 activity is implicated in the pathogenesis of several human cancers, and recent studies indicated that the inhibition of LSD1 is a promising therapeutic strategy for acute myeloid leukemia (AML). Decitabine and azacitidine, FDA-approved agents for the treatment of AML and myelodysplastic syndrome, alter global DNA methylation by inhibiting DNA methyltransferases (DNMTs), resulting in apoptosis and differentiation. Since it is reported that LSD1 directly demethylates and stabilizes DNMT1 by protection from degradation, we hypothesized that an LSD1 inhibitor could augment anti-AML activity of DNMT inhibitors. Previously, we demonstrated that TPC-144, a novel reversible LSD1 inhibitor, was efficacious in preclinical models of AML. Here, we report the activity of TPC-144 in combination with decitabine in cell-based and in vivo studies. Material and Methods: In vitro studies on growth inhibition and apoptosis were conducted using AML cell lines by measuring cellular ATP content (Cell-Titer Glo, Promega) and sub-G1 population, respectively. For expression changes of surface markers, cells were stained with specific antibodies (BD Pharmingen) and analyzed by flow cytometry. mRNA levels of LSD1 target genes were quantitated by real-time PCR. LINE-1 methylation levels were evaluated in Global DNA Methylation-LINE-1 Kit (Active Motif). In vivo efficacy was evaluated in human AML xenograft models in SCID mice. TPC-144 and decitabine was administered orally or intraperitoneally, respectively. Results: The combination of TPC-144 and decitabine showed synergistic growth inhibition of human AML cells in vitro. TPC-144 enhanced apoptosis induction in combination with low-dose decitabine at the concentrations at which TPC-144 decreased DNMT1 protein levels and LINE-1 methylation levels. Combination treatment strongly increased expression of surface markers of differentiation such as CD86 and CD11b, compared with single-agent treatment. In AML xenograft models, the combination of TPC-144 and decitabine was tolerated without signs of hematopoietic toxicities, and demonstrated curative antitumor efficacy compared with each inhibitor alone in a decitabine-sensitive xenograft model. Importantly, the combination treatment of TPC-144 and decitabine exhibited strong antitumor efficacy even in a decitabine-insensitive AML model. Conclusions: TPC-144, a novel potent and selective reversible LSD1 inhibitor, demonstrated synergistic antitumor efficacy in combination with decitabine in human AML cell lines and xenograft models. This combination was well tolerated and showed strong efficacy not only in decitabine-sensitive but also in decitabine-insensitive AML models. Therefore, combination of TPC-144 and decitabine could enhance their therapeutic value for the treatment of AML patients. Citation Format: Akiko Osada, Aki Kawagishi, Ryo Hatanaka, Takumitsu Machida, Kenjiro Ito, Satoshi Yamashita, Takahiro Ogawa, Tadashi Imaoka, Kenichi Matsuo, Teruhiro Utsugi, Yoshikazu Iwasawa. Combination of TPC-144, a reversible LSD1 inhibitor, and a hypomethylating agent resulted in synergistic antitumor efficacy in preclinical models of AML [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A169.

Identification of downstream metastasis-associated target genes regulated by LSD1 in colon cancer cells.

PurposeThis study aims to identify downstream target genes regulated by lysine-specific demethylase 1 (LSD1) in colon cancer cells and investigate the molecular mechanisms of LSD1 influencing invasion and metastasis of colon cancer.MethodWe obtained the expression changes of downstream target genes regulated by small-interfering RNA-LSD1 and LSD1-overexpression via gene expression profiling in two human colon cancer cell lines. An Affymetrix Human Transcriptome Array 2.0 was used to identify differentially expressed genes (DEGs). We screened out LSD1-target gene associated with proliferation, metastasis, and invasion from DEGs via Gene Ontology and Pathway Studio. Subsequently, four key genes (CABYR, FOXF2, TLE4, and CDH1) were computationally predicted as metastasis-related LSD1-target genes. ChIp-PCR was applied after RT-PCR and Western blot validations to detect the occupancy of LSD1-target gene promoter-bound LSD1.ResultA total of 3633 DEGs were significantly upregulated, and 4642 DEGs were downregulated in LSD1-silenced SW620 cells. A total of 4047 DEGs and 4240 DEGs were upregulated and downregulated in LSD1-overexpressed HT-29 cells, respectively. RT-PCR and Western blot validated the microarray analysis results. ChIP assay results demonstrated that LSD1 might be negative regulators for target genes CABYR and CDH1. The expression level of LSD1 is negatively correlated with mono- and dimethylation of histone H3 lysine4(H3K4) at LSD1- target gene promoter region. No significant mono-methylation and dimethylation of H3 lysine9 methylation was detected at the promoter region of CABYR and CDH1.ConclusionLSD1- depletion contributed to the upregulation of CABYR and CDH1 through enhancing the dimethylation of H3K4 at the LSD1-target genes promoter. LSD1- overexpression mediated the downregulation of CABYR and CDH1expression through decreasing the mono- and dimethylation of H3K4 at LSD1-target gene promoter in colon cancer cells. CABYR and CDH1 might be potential LSD1-target genes in colon carcinogenesis.

The dual role of LSD1 and HDAC3 in STAT5-dependent transcription is determined by protein interactions, binding affinities, motifs and genomic positions.

STAT5 interacts with other factors to control transcription, and the mechanism of regulation is of interest as constitutive active STAT5 has been reported in malignancies. Here, LSD1 and HDAC3 were identified as novel STAT5a interacting partners in pro-B cells. Characterization of STAT5a, LSD1 and HDAC3 target genes by ChIP-seq and RNA-seq revealed gene subsets regulated by independent or combined action of the factors and LSD1/HDAC3 to play dual role in their activation or repression. Genes bound by STAT5a alone or in combination with weakly associated LSD1 or HDAC3 were enriched for the canonical STAT5a GAS motif, and such binding induced activation or repression. Strong STAT5 binding was seen more frequently in intergenic regions, which might function as distal enhancer elements. Groups of genes bound weaker by STAT5a and stronger by LSD1/HDAC3 showed an absence of the GAS motif, and were differentially regulated based on their genomic binding localization and binding affinities. These genes exhibited increased binding frequency in promoters, and in conjunction with the absence of GAS sites, the data indicate a requirement for stabilization by additional factors, which might recruit LSD1/HDAC3. Our study describes an interaction network of STAT5a/LSD1/HDAC3 and a dual function of LSD1/HDAC3 on STAT5-dependent transcription, defined by protein–protein interactions, genomic binding localization/affinity and motifs.

Abstract 4701: A phenotypic cell-based screen to identify novel potential epigenetic anti-cancer drugs from natural compounds

Abstract Epigenetic aberrations such as DNA hypermethylation and repressive chromatin are validated targets for cancer chemotherapy. Since epigenetic modifications are reversible, the goal of epigenetic therapy is to reverse the abnormal alternations in cancer cells and induce tumor suppressor genes reactivation, leading to cancer cell differentiation and cell death. Thus, epigenetic enzymes are attractive drug targets in the field of drug discovery. Many known anti-cancer drugs are derived from natural compounds and there have been reports of natural compounds modulating epigenetic activity. Therefore, it would be of interest to screen natural compounds as potential epigenetic drugs. In an effort to identify novel targets that can reactivate hypermethylated silenced genes, our lab developed a phenotypic-based system, YB5. YB5 is a colon cancer cell line generated by stably transfecting SW48 cells with a vector containing GFP driven by a methylated and silenced CMV promoter. GFP re-expression can be achieved by known epigenetic drugs that lead to demethylation or induce active chromatin marks in the CMV promoter. By screening an NDL-3040 library and grouping the molecules based on chemical structures, we were able to identify three main drug classes. The Moulder Center then synthesized 60 new analogs based on class #1's lead's structure and 15 are positive in the YB5 system. The most potent analog can induce 10% GFP+ cells upon 500nM treatment. All the positive hits can also be validated in two other cancer cells (MCF7 and HCT116). Consistent with GFP reactivation, endogenous hypermethylated genes can be reactivated upon drug treatment. Also, GFP positive cells show higher endogenous gene reactivation than unsorted and GFP negative cells. By performing RNA-seq analysis upon class #1 top lead treatment followed by connectivity mapping, we identified X as the class #1 drug target. The on-target effect can be validated by using other selective X inhibitors as well as a dominant negative X construct. Consistent with drug inhibition, dominant-negative X can also reactivate drug targeted hypermethylated genes. Proliferation assays showed differential sensitivities of a panel of colon cancer cell lines compared to normal cells. These drugs can also lead to G2/M arrest and GFP positive cells are more likely to be arrested than GFP negative cells. Besides class #1 drugs, a novel class of LSD1 inhibitors was identified and the most active drug can induce 15% GFP at 5uM. Consistent with LSD1 inhibition, many known LSD1 target genes can also be upregulated. Like known LSD1 inhibitors, these compounds significantly inhibited proliferation of AML cells. We also identified some known natural compounds that have epigenetic activities, including arsenic trioxide, cardiac glycosides, and toyocamycin. Thus, many novel epigenetic drug classes derived from natural compounds were identified and can be developed by targeting silenced gene expression. Citation Format: Hanghang Zhang, Noël J.-M Raynal, Takahiro Sato, Yasuyuki Okamoto, Judit Garriga, George Morton, Wayne Childers, Marlene A. Jacobson, Stephen B. Baylin, Xavier Graña, Magid Abou-Gharbia, Jean-Pierre J. Issa. A phenotypic cell-based screen to identify novel potential epigenetic anti-cancer drugs from natural compounds. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4701.

Abstract 3518: Growth inhibition of SCLC cell lines by treatment with LSD1 inhibitors is associated with modulation of neuroendocrine pathways

Abstract Small cell lung cancer (SCLC) is an aggressive malignancy with a high propensity for early metastasis. Response rates to first-line chemotherapy are typically high, but short lived. We describe an epigenetic-based mechanism for targeting SCLC using inhibitors for the LSD1 histone lysine demethylase. Mechanistically reversible and irreversible LSD1 inhibitors (GSK690 and OG-86) demonstrate induction of either cell cycle arrest or apoptosis in 54% of SCLC cell lines (12/22) that becomes apparent upon continuous treatment for 7-to-10 days. Maximal rates of growth inhibition in sensitive cell lines vary from 50-to-95% and plateaus between 18-to-21 days of LSD1 inhibitor treatment. Thus, heterogeneous sensitivity to LSD1 inhibitors exists between cell line models as well as within subpopulations of cells in the same cell line. To understand the mechanisms underlying LSD1 inhibitor activity in SCLC we have performed RNA-seq and ChIP-seq experiments coupled with bioinformatic analysis of expression signatures in sensitive and resistant models. Our data indicate that although LSD1 is over-expressed in SCLC cell lines and patient samples relative to non-small cell lung cancers, high LSD1 expression does not predict sensitivity to LSD1 inhibitors. Pathway analysis demonstrates that LSD1 inhibition modulates the expression of genes involved in cell adhesion and axon guidance including members of the Ephrin and Semaphorins families. At LSD1 target genes, we demonstrate site-specific H3K4me2 histone methylation changes overlapping LSD1 binding sites, however no global changes in H3K4me2 were observed. Interestingly we find morphological and cell adhesion changes in sensitive cell lines that coincide with expression changes in markers of neuroendocrine differentiation of SCLC such as GRP, NCAM, and NEUROD1. Based on these data, we propose a model that LSD1 inhibition modulates the neuroendocrine differentiation program of SCLC cells promoting tumor inhibition in sensitive SCLC models. Citation Format: Thomas A. Paul, Shikhar Sharma, Jill Hallin, Tao Xie, Timothy Nichols, Mike Greig, James Hardwick, Martin Wythes, Dominique Verhelle. Growth inhibition of SCLC cell lines by treatment with LSD1 inhibitors is associated with modulation of neuroendocrine pathways. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3518. doi:10.1158/1538-7445.AM2015-3518

Abstract 3510: Novel lysine demethylase LSD1 inhibitors promote differentiation and apoptosis of glioma stem cells

Abstract Glioblastoma (GBM) is the most common and devastating primary tumor of the central nervous system with dismal prognosis. GBM development is a multistep process, involving alterations in genetic and epigenetic mechanisms. Understanding the mechanisms and enzymes that promote epigenetic changes in GBM are urgently needed to identify novel therapeutic targets. Recent studies demonstrated that Glioma Stem Cells (GSCs) play central roles in GBM development and responsible for tumor propagation, invasion and chemo/radiation resistance. GSCs elimination is critical for development of efficient therapeutic strategies for GBM. Recently, we showed that lysine demethylase LSD1 is overexpressed in GBM and its inhibition reduces proliferation of GBM cells. In this study, we tested whether, LSD1 modulates GSCs differentiation and novel LSD1 inhibitors (NCL-1 and NCD-38) promote differentiation and apoptosis of GSCs. We have isolated GSCs using well established stem markers (CD133 and CD15) from two established and two patient derived GBM cells. Functionality of GSCs was established using self-renewal assays, and by profiling stem cell marker expression. Western analysis of purified GSCs showed that LSD1 is highly expressed in purified GSCs. Differentiation of GSCs resulted in decreased expression of LSD1. Down regulation of LSD1 expression using lentiviral LSD1-shRNA or by pharmacological inhibition (NCL-1 and NCD-38) significantly reduced the proliferation, neurosphere formation and self-renewal ability of GSCs with little or no effect on normal astrocytes and differentiated cells. Accordingly, treatment of GSCs with LSD1 inhibitors resulted in a dramatic reduction in the expression of stemness markers and promoted induction of differentiation markers. RNA sequencing analysis of GSCs treated with or without LSD1 inhibitors, revealed that LSD1 inhibitors modulate several pathways related to stem ness, differentiation and apoptosis. The expression of top five genes and three pathways were validated using q-RT-PCR, and western blot analysis. Chromatin Immunoprecipitation assays confirmed significant alternations in epigenetic markers at LSD1 target genes. Further, LSD1 inhibitors significantly reduced the growth of GSC driven tumors in vivo. Taken together, these results provided the evidence that induction of GSCs differentiation and apoptosis following LSD1 inhibition as a promising approach to eradicate GSCs. Citation Format: Gangadhara R. Sareddy, Takayoshi Suzuki, Andrew J. Brenner, Ratna K. Vadlamdui. Novel lysine demethylase LSD1 inhibitors promote differentiation and apoptosis of glioma stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3510. doi:10.1158/1538-7445.AM2015-3510

Abstract 3519: A phenotypic screen to discover novel epigenetic anticancer drugs from natural compounds

Abstract Epigenetics is the study of heritable changes in gene expression that are not caused by changes in DNA sequence. Since epigenetic modifications are reversible, the goal of epigenetic therapy is to reverse the abnormal alternations in cancer cells and induce tumor suppressor genes reactivation, leading to cancer cell differentiation and cell death. Thus, epigenetic enzymes are attractive drug targets in the field of drug discovery. Many known anti-cancer drugs are derived from natural compounds and there have been reports of natural compounds modulating epigenetic activity. Therefore, it would be of interest to screen natural compounds as potential epigenetic drugs. We screened 3040 natural compounds and derivatives by measuring GFP expression in the YB5 cell line, a colon cancer cell line generated by stably transfecting SW48 cells with a vector containing GFP driven by a methylated and silenced CMV promoter. GFP re-expression can be achieved by known epigenetic drugs that lead to demethylation or induce active chromatin marks in the CMV promoter. After 24hr treatment, FACS analysis was used to check the GFP expression levels. After the primary screening (average Z’ factor = 0.6), we set a stringent criterion that GFP induction value should be more than the average of all drugs mean +3 standard deviations in order to be considered as positive. 33 hits were positive (positive rate = 1.1%) among which 18 hits were validated through 24hr dose curves, fluorescence microscopy and qPCR. We then grouped the positive hits based on chemical structures. Two classes were selected for further studies. For class#1 compounds, the most active drug induced 20% GFP at 10uM. For this class, we found no effects on DNA methylation, HDAC activity or effects on known histone methyltransferases/demethylases (HMT/HDM) using biochemistry-based assays. Global histone acetylation level was determined using mass spectrometry and we saw upregulation of H4K16ac levels. Proliferation assays showed differential sensitivity of a panel of colon cancer cell lines compared to normal cells (IMR90). These drugs also reactivated two endogenously hypermethylated genes (CDH13 and MGMT). For class#2 compounds, the most active drug activated 15% GFP at 10uM. By using a HMT/HDM biochemistry assay panel, class #2 was determined to inhibit LSD1. qPCR analysis demonstrated upregulation of endogenous LSD1 target genes. Furthermore, like known LSD1 inhibitors, these compounds significantly inhibited cell proliferation of AML cells. Both drug classes can synergize with decitabine (a DNMT inhibitor) to reactivate different tumor suppressor genes. Thus, two novel epigenetic drug classes derived from natural compounds were discovered, with activities on LSD1 and H4K16 acetylation. Citation Format: Hanghang Zhang, Noël Raynal, Takahiro Sato, Yasuyuki Okamoto, Ryan Henry, Andrew J. Andrews, George Morton, Wayne Childers, Marlene A. Jacobson, Magid Abou-Gharbia, Jean-Pierre J. Issa. A phenotypic screen to discover novel epigenetic anticancer drugs from natural compounds. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3519. doi:10.1158/1538-7445.AM2015-3519

SUV39H2 methylates and stabilizes LSD1 by inhibiting polyubiquitination in human cancer cells.

LSD1 is a histone lysine demethylase, which is highly expressed in multiple types of human cancer. Although its roles in transcriptional regulation have been well-studied, functional regulation of LSD1 by post-translational modifications still remains unknown. Here, we demonstrate that the histone lysine methyltransferase SUV39H2 trimethylated LSD1 on lysine 322. Knockdown of SUV39H2 resulted in a decrease of LSD1 protein even though the mRNA levels were unchanged. SUV39H2-induced LSD1 methylation suppresses LSD1 polyubiquitination and subsequent degradation. In addition, we also observed indirect effect of SUV39H2 overexpression on LSD1-target genes. Our results reveal the regulatory mechanism of LSD1 protein through its lysine methylation by SUV39H2 in human cancer cells.

Lsd1 Restricts the Number of Germline Stem Cells by Regulating Multiple Targets in Escort Cells

Specialized microenvironments called niches regulate tissue homeostasis by controlling the balance between stem cell self-renewal and the differentiation of stem cell daughters. However the mechanisms that govern the formation, size and signaling of in vivo niches remain poorly understood. Loss of the highly conserved histone demethylase Lsd1 in Drosophila escort cells results in increased BMP signaling outside the cap cell niche and an expanded germline stem cell (GSC) phenotype. Here we present evidence that loss of Lsd1 also results in gradual changes in escort cell morphology and their eventual death. To better characterize the function of Lsd1 in different cell populations within the ovary, we performed Chromatin immunoprecipitation coupled with massive parallel sequencing (ChIP-seq). This analysis shows that Lsd1 associates with a surprisingly limited number of sites in escort cells and fewer, and often, different sites in cap cells. These findings indicate that Lsd1 exhibits highly selective binding that depends greatly on specific cellular contexts. Lsd1 does not directly target the dpp locus in escort cells. Instead, Lsd1 regulates engrailed expression and disruption of engrailed and its putative downstream target hedgehog suppress the Lsd1 mutant phenotype. Interestingly, over-expression of engrailed, but not hedgehog, results in an expansion of GSC cells, marked by the expansion of BMP signaling. Knockdown of other potential direct Lsd1 target genes, not obviously linked to BMP signaling, also partially suppresses the Lsd1 mutant phenotype. These results suggest that Lsd1 restricts the number of GSC-like cells by regulating a diverse group of genes and provide further evidence that escort cell function must be carefully controlled during development and adulthood to ensure proper germline differentiation.

Involvement of Histone Demethylase LSD1 in Short-Time-Scale Gene Expression Changes during Cell Cycle Progression in Embryonic Stem Cells

The histone demethylase LSD1, a component of the CoREST (corepressor for element 1-silencing transcription factor) corepressor complex, plays an important role in the downregulation of gene expression during development. However, the activities of LSD1 in mediating short-time-scale gene expression changes have not been well understood. To reveal the mechanisms underlying these two distinct functions of LSD1, we performed genome-wide mapping and cellular localization studies of LSD1 and its dimethylated histone 3 lysine 4 (substrate H3K4me2) in mouse embryonic stem cells (ES cells). Our results showed an extensive overlap between the LSD1 and H3K4me2 genomic regions and a correlation between the genomic levels of LSD1/H3K4me2 and gene expression, including many highly expressed ES cell genes. LSD1 is recruited to the chromatin of cells in the G(1)/S/G(2) phases and is displaced from the chromatin of M-phase cells, suggesting that LSD1 or H3K4me2 alternatively occupies LSD1 genomic regions during cell cycle progression. LSD1 knockdown by RNA interference or its displacement from the chromatin by antineoplastic agents caused an increase in the levels of a subset of LSD1 target genes. Taken together, these results suggest that cell cycle-dependent association and dissociation of LSD1 with chromatin mediates short-time-scale gene expression changes during embryonic stem cell cycle progression.

FAD-dependent lysine-specific demethylase-1 regulates cellular energy expenditure

Environmental factors such as nutritional state may act on the epigenome that consequently contributes to the metabolic adaptation of cells and the organisms. The lysine-specific demethylase-1 (LSD1) is a unique nuclear protein that utilizes flavin adenosine dinucleotide (FAD) as a cofactor. Here we show that LSD1 epigenetically regulates energy-expenditure genes in adipocytes depending on the cellular FAD availability. We find that the loss of LSD1 function, either by short interfering RNA or by selective inhibitors in adipocytes, induces a number of regulators of energy expenditure and mitochondrial metabolism such as PPARγ coactivator-1α resulting in the activation of mitochondrial respiration. In the adipose tissues from mice on a high-fat diet, expression of LSD1-target genes is reduced, compared with that in tissues from mice on a normal diet, which can be reverted by suppressing LSD1 function. Our data suggest a novel mechanism where LSD1 regulates cellular energy balance through coupling with cellular FAD biosynthesis.

Recognition of unmethylated histone H3 lysine 4 links BHC80 to LSD1-mediated gene repression

Histone methylation is crucial for regulating chromatin structure, gene transcription and the epigenetic state of the cell. LSD1 is a lysine-specific histone demethylase that represses transcription by demethylating histone H3 on lysine 4 (ref. 1). The LSD1 complex contains a number of proteins, all of which have been assigned roles in events upstream of LSD1-mediated demethylation apart from BHC80 (also known as PHF21A), a plant homeodomain (PHD) finger-containing protein. Here we report that, in contrast to the PHD fingers of the bromodomain PHD finger transcription factor (BPTF) and inhibitor of growth family 2 (ING2), which bind methylated H3K4 (H3K4me3), the PHD finger of BHC80 binds unmethylated H3K4 (H3K4me0), and this interaction is specifically abrogated by methylation of H3K4. The crystal structure of the PHD finger of BHC80 bound to an unmodified H3 peptide has revealed the structural basis of the recognition of H3K4me0. Knockdown of BHC80 by RNA inhibition results in the de-repression of LSD1 target genes, and this repression is restored by the reintroduction of wild-type BHC80 but not by a PHD-finger mutant that cannot bind H3. Chromatin immunoprecipitation showed that BHC80 and LSD1 depend reciprocally on one another to associate with chromatin. These findings couple the function of BHC80 to that of LSD1, and indicate that unmodified H3K4 is part of the 'histone code'. They further raise the possibility that the generation and recognition of the unmodified state on histone tails in general might be just as crucial as post-translational modifications of histone for chromatin and transcriptional regulation.

Regulation of LSD1 Histone Demethylase Activity by Its Associated Factors

LSD1 is a recently identified human lysine (K)-specific histone demethylase. LSD1 is associated with HDAC1/2; CoREST, a SANT domain-containing corepressor; and BHC80, a PHD domain-containing protein, among others. We show that CoREST endows LSD1 with the ability to demethylate nucleosomal substrates and that it protects LSD1 from proteasomal degradation in vivo. We find hyperacetylated nucleosomes less susceptible to CoREST/LSD1-mediated demethylation, suggesting that hypoacetylated nucleosomes may be the preferred physiological substrates. This raises the possibility that histone deacetylases and LSD1 may collaborate to generate a repressive chromatin environment. Consistent with this model, TSA treatment results in derepression of LSD1 target genes. While CoREST positively regulates LSD1 function, BHC80 inhibits CoREST/LSD1-mediated demethylation in vitro and may therefore confer negative regulation. Taken together, these findings suggest that LSD1-mediated histone demethylation is regulated dynamically in vivo. This is expected to have profound effects on gene expression under both physiological and pathological conditions.