scholarly journals Pharmacological Inhibitors of LSD1 Promote Differentiation of Myeloid Leukemia Cells through a Mechanism Independent of Histone Demethylation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 267-267 ◽  
Author(s):  
James T Lynch ◽  
Gary J Spencer ◽  
William J Harris ◽  
Alba Maiques-Díaz ◽  
Filippo Ciceri ◽  
...  

Abstract Lysine Specific Demethylase 1 (LSD1 or KDM1A) is one of a number of epigenetic regulators which have recently emerged as candidate therapeutic targets in acute myeloid leukaemia (AML). It is a flavin adenine dinucleotide (FAD) dependent homolog of the amine oxidase family with an ability to demethylate monomethyl or dimethyl lysine 4 (K4) of histone H3, in addition to other substrates. Pharmacological inhibitors of LSD1 such as the tranylcypromine derivatives have already commenced evaluation in early phase clinical trials. While it has been widely assumed that these compounds promote differentiation of AML cells through inhibition of the demethylase activity of LSD1, the precise mechanisms by which LSD1 inhibitors function has not yet been determined. If changes in histone methylation are a central and critical mediator of the effects of LSD1 inhibitors in promoting AML cell differentiation, it would be expected that global changes in transcription would be tightly linked temporally to changes in histone methylation following drug treatment of cells. Through RNA sequencing and ChIP sequencing experiments performed in human THP1 AML cells treated for 24 hours with a potent and specific tranylcypromine-derivative LSD1 inhibitor (ORY86, trans-N-((2-methoxypyridin-3-yl)methyl)-2-phenylcyclopropan-1-amine), we have established that wholescale up regulation of a myeloid differentiation transcription programme occurs in the absence of any significant genome-wide changes in mono- and di-methyl H3K4 and H3K9 (which are key enzymatic targets of LSD1). Thus LSD1 inhibitor-induced up regulation of myeloid differentiation gene expression is not downstream of changes in histone methylation. We further demonstrated that non-enzymatic functions of LSD1 are essential in AML cells by expressing either wild-type (WT) or catalytically inactive LSD1 (K661A) in LSD1 knockdown (KD) THP1 cells. While LSD1 KD cells exhibit myeloid differentiation and loss of clonogenic potential, both the WT and mutant versions of LSD1 were able to rescue the in vitro clonogenic potential of KD cells to an equivalent extent. Thus the histone demethylase activity of LSD1 is not required to sustain AML blasts in an undifferentiated state. Comparison of the transcriptional consequences of LSD1 inhibition with the transcriptional consequences of transcription factor knockdown in THP1 AML cells using GSEA revealed that pharmacological inhibition of LSD1 mimics depletion of GFI1. Immunoprecipitation experiments confirmed the previously described physical association of GFI1 with LSD1. Critically, the physical interactions of LSD1 with GFI1 was reversed by pharmacological inhibition of LSD1 with ORY86. Furthermore, in ChIP sequencing experiments drug treatment led to dissociation of LSD1 from promoters and enhancers. By contrast, there was no disruption of the endogenous level interaction of LSD1 with RCOR1, HDAC1 and HDAC2 (i.e. the CoREST complex) following drug treatment. To determine whether the inhibitor-induced separation of LSD1 from GFI1 is required for induction of myeloid differentiation by ORY86, we performed experiments using a GFI1-LSD1 fusion construct expressed in THP1 cells under the control of a doxycycline-regulated promoter. This construct tethers LSD1 directly to the transcription factor and circumvents any drug induced physical separation. THP1 cells expressing GFI1-LSD1 were drug resistant (as determined by immunophenotyping and clonogenic potential), in contrast to control cells expressing GFI1, LSD1 or an empty vector in the same inducible system. Thus, drug-induced physical separation of GFI1 from LSD1 is required for THP1 AML cells to undergo differentiation. Our data support a model whereby the physical association of LSD1 with transcription factors such as GFI1 is essential to maintain the differentiation block in AML. Unexpectedly, tranylcypromine-derivative inhibitors target this novel scaffolding function of LSD1, rather than its histone demethylase activity, to promote differentiation of AML cells. Disclosures Lynch: Astra Zeneca: Employment. Ciceri:Oryzon Genomics: Employment. Somervaille:Oryzon Genomics: Research Funding; Imago Biosciences: Consultancy.

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Hayden AM Hatch ◽  
Helen M Belalcazar ◽  
Owen J Marshall ◽  
Julie Secombe

Mutations in the lysine demethylase 5 (KDM5) family of transcriptional regulators are associated with intellectual disability, yet little is known regarding their spatiotemporal requirements or neurodevelopmental contributions. Utilizing the mushroom body (MB), a major learning and memory center within the Drosophila brain, we demonstrate that KDM5 is required within ganglion mother cells and immature neurons for proper axogenesis. Moreover, the mechanism by which KDM5 functions in this context is independent of its canonical histone demethylase activity. Using in vivo transcriptional and binding analyses, we identify a network of genes directly regulated by KDM5 that are critical modulators of neurodevelopment. We find that KDM5 directly regulates the expression of prospero, a transcription factor that we demonstrate is essential for MB morphogenesis. Prospero functions downstream of KDM5 and binds to approximately half of KDM5-regulated genes. Together, our data provide evidence for a KDM5-Prospero transcriptional axis that is essential for proper MB development.


Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1061-1061
Author(s):  
Anetta Ptasinska ◽  
Salam Assi ◽  
Natalia Martinez-Soria ◽  
Maria Rosaria Imperato ◽  
Jason Piper ◽  
...  

Abstract One of the best-characterized chromosomal rearrangements found in AML is the t(8;21) translocation which accounts for approximately 10% of all AMLs and fuses the DNA-binding domain of the hematopoietic master regulator RUNX1 to the almost the entire ETO protein. The resulting RUNX1/ETO fusion protein lacks the transactivation domain of RUNX1 resulting in major differences in the biological activities of RUNX1 and RUNX1/ETO. RUNX1 normally recruits transcriptional activators and binds to DNA as a heterodimer with CBFb which is important for high-affinity DNA binding. The RUNX1/ETO fusion protein also interacts with CBFb but functions as a RUNX1/ETO tetramer, and like ETO itself, it also interacts with NCOR and SIN3A co-repressors. The leukemogenic fusion protein RUNX1/ETO forms a complex with other transcription factors and the binding of this complex to RUNX1 target genes causes a block in myeloid differentiation. Here we investigate the dynamic changes in global transcription factor binding patterns that occur immediately following depletion of RUNX1/ETO. To that end, we combined digital footprinting, ChIP-sequencing for multiple factors and transcriptome analysis to identify the core transcriptional network of t(8;21) AML cells, and then characterized changes in these networks upon RUNX1/ETO knockdown. These analyses revealed a dynamic equilibrium between RUNX1/ETO and RUNX1 complexes competing for the same genomic sites. We show by sequential ChIP that both complexes have similar transcription factor compositions, but differ in their preference for the recruitment of co-activators and co-repressors. Using a novel digital DNaseI footprinting approach we show that both t(8;21)-positive cell lines and patient-derived primary AML cells share the same pattern of binding site occupancy. Within this core transcriptional network, RUNX1/ETO-bound loci are predominantly associated with gene repression. Furthermore, loss of RUNX1/ETO establishes a new differentiation-associated transcriptional network dominated by de novo binding of C/EBPa resulting from the up-regulation of CEBPA gene expression. Our results demonstrate that the block in myeloid differentiation in t(8;21) is caused by the dynamic interference of RUNX1/ETO with cis-regulatory elements destined to bind alternate factor assemblies during myeloid differentiation including both RUNX1 and C/EBPa. Disclosures No relevant conflicts of interest to declare.


2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 371.1-371
Author(s):  
A. Koltakova ◽  
A. Lila ◽  
L. P. Ananyeva ◽  
A. Fedenko

Background:Pts with cancer may have MD that can be caused by neoplastic/paraneoplastic disease, rheumatic diseases or be induced by anticancer drug treatment. There is no data about MD influence on the QoL of cancer patients. The EORTC QoL questionnaire (QLQ)-C30 is a valid questionnaire designed to assess different aspects (Global health (GH), Functional (FS) and symptoms (SS) scales) that define the QoL of cancer patients [1].Objectives:The objective of the study was to assess the impact of drug induced and other types of MD on the QoL of cancer patients that received anticancer drug treatment by using of EORTC QLQ-C30 v3.0.Methods:The sampling of 123 pts (M/F – 40/83; mean age 54.4±12.8) with breast (32,5%), gastrointestinal (17%), ovary (8%), lung (7%) and other cancer was observed by rheumatologist in the oncology outpatient clinic. All pts received anticancer drug treatment: chemotherapy (104 pts), target therapy (16 pts) checkpoint-inhibitors (14 pts), hormone therapy (13 pts) in different combinations. 102(82.9%) of 123pts had MD include arthritis (12 pts), synovitis (5 pts), arthralgia (66 pts), periarthritis (34 pts), osteodynia (13 pts). There were 58 pts (group 1; M/F – 14/44; mean age 52.5±12.2) with anticancer drug treatment induced MD and 44 pts (group 2; M/F – 16/27; mean age 57.6±13.5) with other type of MD include 26 pts with skeletal metastasis. The were 21 pts (group 3; M/F – 10/11; mean age 52.9±11.1) without MD. All pts fulfilled EORTC QLQ-C30 v3.0 (tab.1).Table 1.The median [Q1;Q3] of results of GH, SS and SS of EORTC QLQ-C30ScaleSubscaleGroup1Group2Group3GH58.3[50;58]58.3[41.7;83.3]50[50;66.7]FS*Physical functioning73.3[60;86.7]73.3[66.7;86.7]86.7[80;93]Role functioning66.7[66.7;100]83.3[50;100]100[83;100]Emotional functioning83.3[66.7;100]75[66.7;91.7]91.6[83.3;100]Social functioning83.3[66.7;100]83.3[50;100]100[83.3;100]SS*Pain33.3[0;50]16.7[0;33.3]0[0;16.7]*There are only the scores that had got a statistical difference between the groups.Kruskal-Wallis H and post-hoc (Dwass-Steel-Critchlow-Fligner (DSCF) pairwise comparisons) tests for data analysis were performed.Results:A Kruskal-Wallis H test has shown a statistically significant difference in physical (χ2(2)=7.54; p=0.023), role (χ2(2)=9.87; p=0.007), emotion (χ2(2)=7.69; p=0.021) functioning and pain (χ2(2)=8.44; p=0.015) scores between the different groups. A post-hoc test with DSCF pairwise comparisons of median has shown a statistically significant difference between 1 and 3 groups (W=3.904; p=0.016) for physical functioning, between 2 and 3 groups (W=3.35; p=0.004) for role functioning, between 2 and 3 groups (W=4.03; p=0.012) for emotional functioning, between 1 and 3 groups (W=-3.97; p=0.014) for pain scale.Conclusion:The study has shown that MD associated with anticancer drug treatment adversely affected the QoL of cancer patients received anticancer drug treatment by reducing a physical functioning and by increasing pain scores. Presence of other types of MD adversely affect the QoL by reducing emotional and role functioning.References:[1]Aaronson NK,et al.The European Organization for Research and Treatment of Cancer QLQ-C30: a quality-of-life instrument for use in international clinical trials in oncology. J Natl Cancer Inst.1993;85(5):365-376. doi:10.1093/jnci/85.5.365Disclosure of Interests:None declared


Genes ◽  
2021 ◽  
Vol 12 (5) ◽  
pp. 630
Author(s):  
Yongqing Lan ◽  
Meng Li ◽  
Shuangli Mi

Hematopoietic differentiation is a well-orchestrated process by many regulators such as transcription factor and long non-coding RNAs (lncRNAs). However, due to the large number of lncRNAs and the difficulty in determining their roles, the study of lncRNAs is a considerable challenge in hematopoietic differentiation. Here, through gene co-expression network analysis over RNA-seq data generated from representative types of mouse myeloid cells, we obtained a catalog of potential key lncRNAs in the context of mouse myeloid differentiation. Then, employing a widely used in vitro cell model, we screened a novel lncRNA, named Gdal1 (Granulocytic differentiation associated lncRNA 1), from this list and demonstrated that Gdal1 was required for granulocytic differentiation. Furthermore, knockdown of Cebpe, a principal transcription factor of granulocytic differentiation regulation, led to down-regulation of Gdal1, but not vice versa. In addition, expression of genes involved in myeloid differentiation and its regulation, such as Cebpa, were influenced in Gdal1 knockdown cells with differentiation blockage. We thus systematically identified myeloid differentiation associated lncRNAs and substantiated the identification by investigation of one of these lncRNAs on cellular phenotype and gene regulation levels. This study promotes our understanding of the regulation of myeloid differentiation and the characterization of roles of lncRNAs in hematopoietic system.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kylie Hin-Man Mak ◽  
Yuk Man Lam ◽  
Ray Kit Ng

AbstractTrophoblast stem cell (TSC) is crucial to the formation of placenta in mammals. Histone demethylase JMJD2 (also known as KDM4) family proteins have been previously shown to support self-renewal and differentiation of stem cells. However, their roles in the context of the trophoblast lineage remain unclear. Here, we find that knockdown of Jmjd2b resulted in differentiation of TSCs, suggesting an indispensable role of JMJD2B/KDM4B in maintaining the stemness. Through the integration of transcriptome and ChIP-seq profiling data, we show that JMJD2B is associated with a loss of H3K36me3 in a subset of embryonic lineage genes which are marked by H3K9me3 for stable repression. By characterizing the JMJD2B binding motifs and other transcription factor binding datasets, we discover that JMJD2B forms a protein complex with AP-2 family transcription factor TFAP2C and histone demethylase LSD1. The JMJD2B–TFAP2C–LSD1 complex predominantly occupies active gene promoters, whereas the TFAP2C–LSD1 complex is located at putative enhancers, suggesting that these proteins mediate enhancer–promoter interaction for gene regulation. We conclude that JMJD2B is vital to the TSC transcriptional program and safeguards the trophoblast cell fate via distinctive protein interactors and epigenetic targets.


Author(s):  
Tongbin Wu ◽  
Zhengyu Liang ◽  
Zengming Zhang ◽  
Canzhao Liu ◽  
Lunfeng Zhang ◽  
...  

Background: Left ventricular noncompaction cardiomyopathy (LVNC) was discovered half a century ago as a cardiomyopathy with excessive trabeculation and a thin ventricular wall. In the decades since, numerous studies have demonstrated that LVNC primarily impacts left ventricles (LVs), and is often associated with LV dilation and dysfunction. However, owing in part to the lack of suitable mouse models that faithfully mirror the selective LV vulnerability in patients, mechanisms underlying susceptibility of LV to dilation and dysfunction in LVNC remain unknown. Genetic studies have revealed that deletions and mutations in PRDM16 cause LVNC, but previous conditional Prdm16 knockout mouse models do not mirror the LVNC phenotype in patients, and importantly, the underlying molecular mechanisms by which PRDM16 deficiency causes LVNC are still unclear. Methods: Prdm16 cardiomyocyte (CM)-specific knockout ( Prdm16 cKO ) mice were generated and analyzed for cardiac phenotypes. RNA sequencing and ChIP sequencing were performed to identify direct transcriptional targets of PRDM16 in CMs. Single cell RNA sequencing in combination with Spatial Transcriptomics were employed to determine CM identity at single cell level. Results: CM-specific ablation of Prdm16 in mice caused LV-specific dilation and dysfunction, as well as biventricular noncompaction, which fully recapitulated LVNC in patients. Mechanistically, PRDM16 functioned as a compact myocardium-enriched transcription factor, which activated compact myocardial genes while repressing trabecular myocardial genes in LV compact myocardium. Consequently, Prdm16 cKO LV compact myocardial CMs shifted from their normal transcriptomic identity to a transcriptional signature resembling trabecular myocardial CMs and/or neurons. Chamber-specific transcriptional regulation by PRDM16 was in part due to its cooperation with LV-enriched transcription factors Tbx5 and Hand1. Conclusions: These results demonstrate that disruption of proper specification of compact CM may play a key role in the pathogenesis of LVNC. They also shed light on underlying mechanisms of LV-restricted transcriptional program governing LV chamber growth and maturation, providing a tangible explanation for the susceptibility of LV in a subset of LVNC cardiomyopathies.


2016 ◽  
Vol 113 (27) ◽  
pp. E3911-E3920 ◽  
Author(s):  
Eden Kleiman ◽  
Haiqun Jia ◽  
Salvatore Loguercio ◽  
Andrew I. Su ◽  
Ann J. Feeney

Ying Yang 1 (YY1) is a ubiquitously expressed transcription factor shown to be essential for pro–B-cell development. However, the role of YY1 in other B-cell populations has never been investigated. Recent bioinformatics analysis data have implicated YY1 in the germinal center (GC) B-cell transcriptional program. In accord with this prediction, we demonstrated that deletion of YY1 by Cγ1-Cre completely prevented differentiation of GC B cells and plasma cells. To determine if YY1 was also required for the differentiation of other B-cell populations, we deleted YY1 with CD19-Cre and found that all peripheral B-cell subsets, including B1 B cells, require YY1 for their differentiation. Transitional 1 (T1) B cells were the most dependent upon YY1, being sensitive to even a half-dosage of YY1 and also to short-term YY1 deletion by tamoxifen-induced Cre. We show that YY1 exerts its effects, in part, by promoting B-cell survival and proliferation. ChIP-sequencing shows that YY1 predominantly binds to promoters, and pathway analysis of the genes that bind YY1 show enrichment in ribosomal functions, mitochondrial functions such as bioenergetics, and functions related to transcription such as mRNA splicing. By RNA-sequencing analysis of differentially expressed genes, we demonstrated that YY1 normally activates genes involved in mitochondrial bioenergetics, whereas it normally down-regulates genes involved in transcription, mRNA splicing, NF-κB signaling pathways, the AP-1 transcription factor network, chromatin remodeling, cytokine signaling pathways, cell adhesion, and cell proliferation. Our results show the crucial role that YY1 plays in regulating broad general processes throughout all stages of B-cell differentiation.


2021 ◽  
Vol 118 (46) ◽  
pp. e2113579118
Author(s):  
Yuko Hasegawa ◽  
Kevin Struhl

Using a tamoxifen-inducible time-course ChIP-sequencing (ChIP-seq) approach, we show that the ubiquitous transcription factor SP1 has different binding dynamics at its target sites in the human genome. SP1 very rapidly reaches maximal binding levels at some sites, but binding kinetics at other sites is biphasic, with rapid half-maximal binding followed by a considerably slower increase to maximal binding. While ∼70% of SP1 binding sites are located at promoter regions, loci with slow SP1 binding kinetics are enriched in enhancer and Polycomb-repressed regions. Unexpectedly, SP1 sites with fast binding kinetics tend to have higher quality and more copies of the SP1 sequence motif. Different cobinding factors associate near SP1 binding sites depending on their binding kinetics and on their location at promoters or enhancers. For example, NFY and FOS are preferentially associated near promoter-bound SP1 sites with fast binding kinetics, whereas DNA motifs of ETS and homeodomain proteins are preferentially observed at sites with slow binding kinetics. At promoters but not enhancers, proteins involved in sumoylation and PML bodies associate more strongly with slow SP1 binding sites than with the fast binding sites. The speed of SP1 binding is not associated with nucleosome occupancy, and it is not necessarily coupled to higher transcriptional activity. These results with SP1 are in contrast to those of human TBP, indicating that there is no common mechanism affecting transcription factor binding kinetics. The biphasic kinetics at some SP1 target sites suggest the existence of distinct chromatin states at these loci in different cells within the overall population.


Science ◽  
2019 ◽  
Vol 363 (6432) ◽  
pp. 1217-1222 ◽  
Author(s):  
Abhishek A. Chakraborty ◽  
Tuomas Laukka ◽  
Matti Myllykoski ◽  
Alison E. Ringel ◽  
Matthew A. Booker ◽  
...  

Oxygen sensing is central to metazoan biology and has implications for human disease. Mammalian cells express multiple oxygen-dependent enzymes called 2-oxoglutarate (OG)-dependent dioxygenases (2-OGDDs), but they vary in their oxygen affinities and hence their ability to sense oxygen. The 2-OGDD histone demethylases control histone methylation. Hypoxia increases histone methylation, but whether this reflects direct effects on histone demethylases or indirect effects caused by the hypoxic induction of the HIF (hypoxia-inducible factor) transcription factor or the 2-OG antagonist 2-hydroxyglutarate (2-HG) is unclear. Here, we report that hypoxia promotes histone methylation in a HIF- and 2-HG–independent manner. We found that the H3K27 histone demethylase KDM6A/UTX, but not its paralog KDM6B, is oxygen sensitive. KDM6A loss, like hypoxia, prevented H3K27 demethylation and blocked cellular differentiation. Restoring H3K27 methylation homeostasis in hypoxic cells reversed these effects. Thus, oxygen directly affects chromatin regulators to control cell fate.


2019 ◽  
Author(s):  
Arif Harmanci ◽  
Akdes Serin Harmanci ◽  
Jyothishmathi Swaminathan ◽  
Vidya Gopalakrishnan

Abstract Motivation Functional genomics experiments generate genomewide signal profiles that are dense information sources for annotating the regulatory elements. These profiles measure epigenetic activity at the nucleotide resolution and they exhibit distinctive patterns as they fluctuate along the genome. Most notable of these patterns are the valley patterns that are prevalently observed in assays such as ChIP Sequencing and bisulfite sequencing. The genomic positions of valleys pinpoint locations of cis-regulatory elements such as enhancers and insulators. Systematic identification of the valleys provides novel information for delineating the annotation of regulatory elements. Nevertheless, the valleys are not reported by majority of the analysis pipelines. Results We describe EpiSAFARI, a computational method for sensitive detection of valleys from diverse types of epigenetic profiles. EpiSAFARI employs a novel smoothing method for decreasing noise in signal profiles and accounts for technical factors such as sparse signals, mappability, and nucleotide content. In performance comparisons, EpiSAFARI performs favorably in terms of accuracy. The histone modification valleys detected by EpiSAFARI exhibit high conservation, transcription factor binding, and they are enriched in nascent transcription. In addition, the large clusters of histone valleys are found to be enriched at the promoters of the developmentally associated genes. Differential histone valleys exhibit concordance with differential DNase signal at cell line specific valleys. DNA methylation valleys exhibit elevated conservation and high transcription factor binding. Specifically, we observed enriched binding of transcription factors associated with chromatin structure around methyl-valleys. Availability EpiSAFARI is publicly available at https://github.com/harmancilab/EpiSAFARI Supplementary information Supplementary data are available at Bioinformatics online.


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