Inhibition of LSD1 Influences Multiple Mechanisms of Epigenetic Gene Regulation During Terminal Erythroid Maturation

Abstract Abstract 3442 The maturation of a committed erythroid progenitor to a functional red blood cell is a complex process involving significant changes in gene expression during a time of rapid cell division and nuclear condensation. LSD1 (Lysine-Specific Histone Demethylase 1) is a histone H3 lysine 4 (H3K4) and lysine 9 (H3K9) demethylase that plays pivotal role in this process. LSD1 participates in both enhancer and repressor complexes, and facilitates repression of γ-globin by participating in the Direct Repeat Erythroid Complex (Cui, MCB, 2011). LSD1 inhibitors Tranylcypromine (TCP) and Pargyline (PG) are being investigated as potential therapies for the β-globinopathies, however little is known about the broader functional or genomic consequences of LSD1 inhibition on terminal erythroid maturation. Both TCP and PG impair erythroid maturation in Extensively Self Renewing Erythroblasts (ESREs), a primary cell model of terminal erythroid maturation. ESREs are primary cells derived from fetal liver that proliferate extensively in culture, but retain the ability to appropriately mature and enucleate (England, Blood, 2011), making them ideal for functional and genomic studies of terminal erythroid maturation. In untreated or vehicle (DMSO) treated cultures >90% of cells are benzidine positive by day3 of maturation. In contrast, cultures treated with 400um PG, 1um TCP, or 2um TCP were 72, 42, and 33% benzidine positive by maturation day3, respectively. Cells in the TCP-and PG- treated cultures also had morphologic evidence of impaired maturation, with larger nuclei and more basophilic cytoplasm. In addition to its role as a histone demethylase, LSD1 stabilizes DNMT1 (DNA methyltransferase 1; Wang, Nat Genet 2009). We hypothesized that loss of DNA methylation contributes to the maturation impairment seen with LSD1 inhibitors, and that inhibition of DNMTs with decitabine would also impair terminal erythroid maturation. Consistent with this hypothesis, ESREs treated with decitabine demonstrated a dose-dependent impairment of maturation similar to that seen with PG and TCP. To elucidate the molecular mechanisms underlying the maturation impairment in TCP- and PG- treated cultures, levels of H3K4me2 and methylated DNA (5-methyl cytosine, 5-mC) were assessed both globally and at specific loci. An ELISA (Enzyme-linked Immunosorbent Assay) was used to assess global levels of H3K4me2 and 5-mC in vehicle-, PG-, and TCP-treated cultures after 24 hours of maturation. Global levels of H3K4me2 were significantly higher in PG- and TCP- treated samples than control. In maturing cells, there was no significant difference in the level of 5-mC in vehicle- and inhibitor- treated cultures. It is well established, however, that global DNA methylation decreases with erythroid maturation (Seashore, Science, 2011), and a significant decrease in 5-mC occurs in ESREs during the first 24hrs of maturation. As TCP- and vehicle- treated cultures mature differently, the effect of TCP on 5-mC levels was also assessed in self-renewing ESREs at the proerythroblast stage. Unlike maturing cells, TCP-treated proerythroblasts had a significant decrease in 5-mC levels compared to control. Chromatin immunoprecipitation (ChIP) was used to examine the local effects of LSD1 inhibition on H3K4me2 enrichment at erythroid-specific promoters. TCP-treated cultures had non-uniform changes in H3K4me2 enrichment, with levels increased at some promoters (e.g. protein 4.1,εy-globin), but unchanged at others (e.g. β-globin). To further study the relationship between LSD1 inhibition and H3K4me2 levels, ChIP-seq was used to identify LSD1 sites that co-localized with putative enhancers, defined as peaks of H3K4me2 binding > than 1kb from a transcription start site. ChIP-qPCR was used to compare the level of H3K4me2 at 5 validated enhancer-associated LSD1 sites in vehicle- and TCP-treated cells. The effect of TCP was variable, with only 2/5 enhancer-associated LSD1 sites having increased H3K4me2. Lastly, the local effects of inhibitors on 5-mC were examined using a methyl binding domain pulldown coupled with qPCR. In TCP-treated cells, 5-mC levels declined at several loci, most notably at the εy-globin promoter. Taken together, these results suggest that the impaired erythroid maturation associated with LSD1 inhibition results from the perturbation of multiple mechanisms of epigenetic regulation. Disclosures: No relevant conflicts of interest to declare.

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LSD1 Influences Both Histone and DNA Methylation During Terminal Erythroid Maturation

Blood ◽

10.1182/blood.v122.21.3671.3671 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3671-3671

Author(s):

Michael Getman ◽

Jeffrey Malik ◽

James Palis ◽

Laurie A Steiner

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Differentially Expressed Genes ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Histone Demethylase ◽

Differentially Expressed ◽

Enzyme Linked Immunosorbent Assay ◽

Erythroid Progenitor ◽

Erythroid Maturation

Abstract The molecular mechanisms that drive the maturation of a committed erythroid progenitor to a functional red blood cell are incompletely understood. LSD1 (Lysine-Specific Histone Demethylase 1) is a widely expressed histone demethylase that plays an important role in erythroid maturation (Kereyni, elife, 2013). Although LSD1 is important for a number of biologic processes ranging from embryonic development to leukemogenesis, the molecular mechanisms underlying the influence of LSD1 on gene expression are incompletely understood. The goal of our study is to elucidate the molecular mechanisms by which LSD1 regulates erythroid gene expression and influences erythroid maturation. We hypothesize that LSD1 promotes specific patterns of histone and DNA methylation that facilitate gene expression changes necessary for normal erythroid maturation to occur. To address this hypothesis, the functional and molecular consequences of LSD1 knockdown were assessed in Extensively Self Renewing Erythroblasts (ESREs), a non-transformed, karyotypically normal model of terminal erythroid maturation (England, Blood, 2011). Primary fetal liver was cultured in the presence of EPO, SCF, IGF1 and dexamethasone to derive ESREs. The ESREs were capable of extensive ex-vivo expansion, doubling daily at the proerythroblast phase, however when matured, >90% of cells became benzidine positive and >65% enucleated within 3 days. Lentiviral-mediated shRNA was used to knock down LSD1 in expanding ESREs. Imaging flow cytometry done on maturation day 3 demonstrated that the knockdown cells had impairments in multiple facets of maturation, with larger cell and nuclear areas, higher kit expression, and lower rates of enucleation than the scramble control. LSD1 knockdown was also associated with impaired hemoglobin accumulation (78% vs. 95% benzidine positive; p<0.005). Treatment of ESREs with an inhibitor to LSD1 (Tranylcypromine; TCP) resulted in similar abnormalities in cell and nuclear size, kit expression, hemoglobin accumulation, and enucleation (40% vehicle vs.1% TCP). The functional deficits in maturation, including abnormal kit expression and low rates of enucleation, persisted on maturation day 4. To delineate the molecular mechanisms underlying this maturation impairment, RNA-seq was done in LSD1 knockdown and scramble control samples, and 230 differentially expressed genes (FDR<0.01) were identified using cuffdiff (Trapnell, Nat Biotech, 2013). Consistent with LSD1’s role in erythroid maturation, Ingenuity Pathway Analysis identified multiple networks involving hemoglobin synthesis, and GATA1, EPO, and KLF1 were all predicted as upstream regulators (p-values of 8.24e10-11, 7.25 e10-6, and 3.86e10-4, respectively). To better understand how LSD1 influences gene expression, chromatin immunoprecipitation coupled with high throughput sequencing was used to identify sites of H3K4me2 binding in the differentially expressed genes. 214/230 differentially expressed genes were associated with sites of H3K4me2 occupancy. Quantitative ChIP demonstrated that LSD1 inhibition was associated with increases in H3K4me2 levels at a subset of these sites, however consistent with previous studies, global levels of H3K4me2, determined by Enzyme Linked Immunosorbent Assay (ELIZA), did not change significantly. Although it is known that LSD1 demethylates and stabilizes the maintenance DNA methyltransferase DNMT1 (Wang, Nat Genet 2009), the consequences of LSD1 loss on DNA methylation (5-methyl cytosine; 5-mC) have yet to be investigated. To gain a comprehensive understanding of how LSD1 regulates erythroid gene expression, changes in the level of 5-mC were assessed after knockdown or inhibition of LSD1. Global 5-mC levels, determined by ELIZA assay, were ∼30% lower in TCP treated samples than vehicle treated control (p<0.02) and western blot demonstrated a 3-fold decrease in DNMT1 protein in the TCP treated samples. Both methyl binding domain pull-down coupled with quantitative PCR and genome-wide bisulfite sequencing were utilized to assess changes in 5-mC levels in the differentially expressed genes. Loss of LSD1 was associated with significantly lower levels of 5-mC at several differentially expressed, erythroid-specific genes, such as bh1. Taken together, these data support the hypothesis that LSD1 influences both histone and DNA methylation at genes important for erythroid maturation. Disclosures: No relevant conflicts of interest to declare.

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DNMT3A Regulates Hematopoietic Stem Cell Function Via DNA Methylation-Independent Functions

Blood ◽

10.1182/blood-2021-153759 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 24-24

Author(s):

Won Kyun Koh ◽

Hamza Celik ◽

Jacob Tao ◽

Jake Fairchild ◽

Ostap Kukhar ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Dna Methyltransferase ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Ectopic Expression ◽

Wild Type ◽

Self Renewal ◽

Hematopoietic Stem ◽

Independent Functions

Abstract The balance between self-renewal and differentiation of hematopoietic stem cells (HSCs) is strictly regulated to sustain blood production throughout adult life. De novo DNA methyltransferase 3-alpha (DNMT3A) is one of the major epigenetic regulators that is essential for efficient HSC differentiation. DNMT3A mutations are prevalent in myeloid diseases that include acute myeloid leukemia (AML; ~22%) and myelodysplastic syndrome (MDS; ~10%) where they act as initiating events However, the precise molecular mechanisms of how DNMT3A regulates normal hematopoiesis and its mutations prime HSCs for leukemic formation are unclear. Although DNMT3A is described as a DNA methyltransferase enzyme, the lack of consistent correlation between changes in DNA methylation and differential gene expression in Dnmt3a-null HSCs in mouse models, and AML patients with DNMT3A mutations undermine the conventional understanding of DNMT3A's canonical role in hematopoietic cells. Hence, we hypothesized that DNMT3A may have novel functions outside of DNA methylation that regulate HSC fate decisions. To answer this question, we first ectopically expressed GFP-labeled Dnmt3a constructs (wild-type Dnmt3a, Dnmt3aE752A; complete DNA methylation dead, and Dnmt3aR832A; reduced DNA methylation target recognition) and empty vector (negative control) in Dnmt3a-null (Vav-Cre: Dnmt3afl/fl = Dnmt3a-/- in hematopoiesis) bone marrow (BM) cells. The result showed that similar to restoring wild-type Dnmt3a, ectopic expression of Dnmt3aE752A as well as Dnmt3aR832A showed a rescue effect of decreased engraftment of transduced cells in the peripheral blood as well as reduced HSC numbers in the BM. Analysis of DNA methylation by whole-genome bisulfite sequencing (WGBS) in transduced cells showed this phenotypic and functional rescue of the Dnmt3a-/- phenotype occurred in the absence of restored DNA methylation patterns. To study the importance of Dnmt3a-mediated DNA methyltransferase activity in a more physiological system, we generated knock-in mice that have one copy of either wild-type Dnmt3a, Dnmt3aE752A, or Dnmt3aR832A (CAGG-Cre-ER T2 = ER T2-Cre: Dnmt3afl/+, Dnmt3afl/E752A, and Dnmt3afl/R832A) to be compared to the Dnmt3a-null group (ER T2-Cre: Dnmt3afl/-). These mice contain one allele with loxP-flanked Dnmt3a that is deleted by tamoxifen-inducible Cre-mediated recombination and one allele of either wild-type Dnmt3a, Dnmt3aE752A, Dnmt3aR832A, or germline knockout Dnmt3anull. 5-weeks post-tamoxifen (~93% floxed allele recombination), competitive transplantation of 250 phenotypically defined test HSCs against with 2.5x10 5 congenic competitor BM cells was performed. Dnmt3a fl/R832A recipients had higher engraftment (35.6 % +/- 6.1) than Dnmt3afl/+ (28.5% +/- 7.2) and Dnmt3afl/- (10.7% +/- 2.79), while Dnmt3afl/E752A had slightly higherengraftment (12.5% +/- 3) than Dnmt3afl/-. Analysis of the BM 18 weeks post-transplant showed that Dnmt3afl/E752A and Dnmt3afl/R832A HSCs phenocopied the HSC self-renewal potential phenotype of heterozygous Dnmt3a fl/+HSCs (Fig. 1). The absolute count of donor-derived HSCs per mouse after the transplant were: ER T2-Cre control (675.7 +/- 299.3), Dnmt3afl/+ (1870 +/- 961.4), Dnmt3afl/- (3546 +/- 1019), Dnmt3afl/E752A (1130 +/- 362.7), and Dnmt3afl/R832A (1184 +/- 344.5) (mean +/- S.E.M.). While the described clonal expansion of Dnmt3a-null HSCs was observed, HSCs with one copy of full-length Dnmt3a but devoid of its methyltransferase capacity mimicked the heterozygous state rather than the homozygous loss-of-function. This is the first evidence to suggest that DNMT3A potentially regulates HSCs by non-canonical (DNA methylation independent) mechanisms. DNA methylation analysis by WGBS is ongoing to determine if Dnmt3afl/E752A and Dnmt3afl/R832A HSCs show a methylome comparable to Dnmt3a-null HSCs whilst having the functional potential of Dnmt3a-heterozygous HSCs, which will be complemented with other molecular analyses including gene expression. Our study opens new avenues for investigations into the molecular mechanisms of DNMT3A function in HSC biology, which could ultimately benefit clinical practice by identifying new therapeutic approaches for the patients with DNMT3A mutations. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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UTX, a Histone Demethylase, Is Inactivated through Homozygous Deletion, Mutation, and DNA Methylation in Multiple Myeloma.

Blood ◽

10.1182/blood.v114.22.1798.1798 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1798-1798

Author(s):

Brian A Walker ◽

Paola E. Leone ◽

Nicholas J Dickens ◽

Kevin D Boyd ◽

David Gonzalez ◽

...

Keyword(s):

Dna Methylation ◽

Cell Lines ◽

Copy Number ◽

Conflicts Of Interest ◽

Cpg Island ◽

Chromatin Condensation ◽

Methylation Status ◽

Significant Proportion ◽

Histone Demethylase ◽

Homozygous Deletions

Abstract Abstract 1798 Poster Board I-824 Histone modifications are known to mediate transcriptional regulation through changes in chromatin condensation and as such can lead to aberrant transcriptional patterns resulting in malignant transformation. Modulation of chromatin structure via histone modification is becoming recognised as an important pathogenic mechanism in myeloma and has been suggested by the over-expression of MMSET, a histone methyltransferase, by the t(4;14) chromosomal rearrangement. More recently inactivation of UTX, a histone demethylase, has also been suggested to have a role in myeloma pathogenesis and both UTX and MMSET are mediators of transcriptional repression. UTX is inactivated in a number of different cancer cell lines but importantly, mutations and deletions have been detected in myeloma cell lines and we wished to follow up on this observation in uniformly treated clinical cases. UTX is a large gene found on the X chromosome covering 240 kb of genomic DNA and consists of 29 exons encoding a protein with both JmjC-domains and tricopeptide repeats responsible for histone demethylation and polycomb protein interactions. Inactivation of UTX occurs through deletions of individual exons through to large whole gene deletions as well as by mutations scattered throughout the 29 exons. A further mechanism of UTX inactivation which has not been looked for to date is via DNA methylation of the CpG island upstream of the transcriptional start site. We set out to determine the status of UTX in our dataset which includes expression, mapping, and methylation array data from presenting myeloma samples entered into the MRC Myeloma IX clinical trial. The gene expression of UTX was measured on 272 samples using Affymetrix U133 Plus 2.0 arrays and showed that 80% of samples do not express UTX transcripts but using expression quartile analysis we could not detect an effect on overall survival. The mechanism underlying the abrogation of expression was investigated further using the Affymetrix 500K SNP mapping array on a subset of 114 samples to detect copy number alterations. UTX was hemizygously deleted in 21 (42%) female samples and was completely deleted in 1 male sample, at the resolution of the arrays. In order to determine if individual exons were deleted, at a resolution below that detectable by mapping arrays, we performed quantitative PCR coupled with high resolution melting (HRM) analysis using the Rotor-gene Q real-time cycler (Qiagen). Exons were amplified, over 40 cycles, to obtain products of ∼200 bp using LC Green Plus mastermix (Idaho Technologies) in a 10 μl reaction on the Rotor-gene Q with a final HRM step from 72-95 °C with increments of 0.1 °C. Amplification plots combined with the HRM step allows us to identify both homozygous deletions and mutations within the exons. We screened all 114 samples for micro-deletions and mutations and found homozygous deletions in ∼7% of samples and identified a significant proportion of mutations using the HRM method which accounted for a total of ∼10% of gene inactivation. In order to determine if methylation could be responsible for inactivation of the remaining allele we used the Illumina Infinium humanmethylation27 array to study the methylation status at the UTX locus. This array interrogates 27,578 highly informative CpG sites per sample at the single-nucleotide resolution using bisulfite converted DNA. The results of this analysis are presented as an average beta-score where 1.0 is fully methylated and 0 is fully unmethylated. Samples were analyzed using Illumina GenomeStudio and the custom differential methylation algorithm. In samples with a diploid copy number of UTX the methylation signals covered 2 ranges: hemi-methylated (0.35-0.55, n=7) and hyper-methylated (0.73-0.89, n=14). In samples with 1 copy of UTX, which includes all males, there were 3 ranges: hypomethylated (0.08-0.21, n=5), hemi-methylated (0.35-0.51, n=3), and hypermethylated (0.66-0.88, n=48). All of the hypomethylated samples with a single copy of UTX were male, and at least 1 of these samples contained an inactivating exonic deletion resulting in complete loss of function. These data indicate that methylation of the residual allele contributes significantly to the inactivation of UTX along with interstitial deletions and mutations. We will go on to present data on the interaction of UTX with variation at the UTY locus and how this modulates behaviour of the myeloma clone. Disclosures No relevant conflicts of interest to declare.

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Serine Phosphorylation On TAL1 Regulates Its Interaction with Histone Demethylase LSD1.

Blood ◽

10.1182/blood.v114.22.1460.1460 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1460-1460

Author(s):

Ying Li ◽

Xin Hu ◽

River Ybarra ◽

Xueqi Fu ◽

Yi Qiu ◽

...

Keyword(s):

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Erythroid Differentiation ◽

Histone Demethylase ◽

Serine Phosphorylation ◽

Rapid Decline ◽

Erythroid Progenitor ◽

Functional Link ◽

Inhibition Of Proliferation ◽

Interacting Domain

Abstract Abstract 1460 Poster Board I-483 TAL1, originally identified by virtue of its involvement in a T-cell acute lymphoblastic leukemia (T-ALL)-specific chromosomal translocation, is a member of the basic helix-loop-helix (bHLH) family of transcription factors and is required for the development of all hematopoietic cell lineages. TAL1 is a phosphorylated protein and its activities are mediated by the corepressors and coactivators that associate with TAL1. However, the functional link between phosphorylation and the recruitment of co-regulators by TAL1 is currently unknown. We undertook the biochemical purification of the TAL1 containing complexes and showed that TAL1 is associated with histone demethylase complexes containing LSD1, CoREST, HDAC1 and HDAC2. This complex mediates TAL1 directed transcriptional repression during hematopoiesis. The interaction between TAL1 and LSD1 are dynamically regulated and is required for TAL1's function in erythroid differentiation (Proc. Natl. Acad. Sci. USA 106: 10141-10146). To further understand the molecular mechanism that regulates the TAL1 and LSD1 interaction during hematopoiesis, we determined whether TAL1 directly interacts with LSD1 and characterized the domains required for this interaction. TAL1 and its various deletion mutants were tested for their ability to interact with LSD1 in vitro. TAL1 directly interacts with LSD1, and the interacting domain encompasses amino acids 142-185 proximal to the bHLH domain, which contains a serine 172 residue that becomes phosphorylated during transcriptional activation of TAL1. We further mutated serine 172 of TAL1 to Alanine (Ala) or to Aspartic acid (Asp) to mimic unphosphorylated or phosphorylated TAL1, respectively. While the TAL1Ser172Ala mutant remains the interaction with LSD1, TAL1Ser172Asp specifically loses its interaction with LSD1 indicating that serine 172 phosphorylation of TAL1 destabilizes the TAL1 and LSD1 interaction. Given that our previous results indicated that LSD1 inhibits TAL1 mediated erythroid differentiation, to further test whether the activity of TAL1 is mediated through interaction with LSD1, we expressed the TAL1 mutant that deleted the LSD1 interacting domain in erythroid progenitor cells and showed that the deletion of the LSD1 interacting domain of TAL1 lead to a promotion of erythroid differentiation and inhibition of proliferation. Furthermore, consistent with the rapid decline of TAL1-associated LSD1 complex during differentiation, the ChIP and ChIP-seq data showed that H3K4 di- and tri-methylation are enriched at the promoters of TAL1 target genes upon erythroid differentiation. Thus, our data revealed that histone lysine demethylase LSD1 may negatively regulate TAL1-mediated transcription and erythroid differentiation. The results suggest that the dynamic regulation of TAL1-associated LSD1/HDAC1 complex may determine the onset of erythroid differentiation programs. * These authors contribute equally to this work. Disclosures No relevant conflicts of interest to declare.

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DNA Methylation Changes Following 5-Azacitidine Treatment in Patients with Myelodysplastic Syndrome.

Blood ◽

10.1182/blood.v114.22.2791.2791 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2791-2791

Author(s):

Huong Thi Thanh Tran ◽

Hee Nam Kim ◽

Yeo-Kyeoung Kim ◽

Jae-Sook Ahn ◽

Il-Kwon Lee ◽

...

Keyword(s):

Dna Methylation ◽

Tumor Suppressor ◽

Tumor Suppressor Genes ◽

Dna Methyltransferase ◽

Conflicts Of Interest ◽

Control Group ◽

Disease Pathogenesis ◽

Suppressor Genes ◽

Clinical Responses ◽

Dependent Probe

Abstract Abstract 2791 Poster Board II-767 Gene silencing by promoter methylation is as potent as functional inactivating of tumor suppressor genes by mutations. DNA methyltransferase inhibitor, 5-azacytidine (AC) and 5-aza-2 -deoxycitidine (DAC), which is proved to be effective in myelodysplastic syndromes (MDS) can induce re-expression in cancer; however their mechanism remains controversial. 25 tumor suppressor genes by MS-MLPA (methylation-specific multiplex ligation-dependent probe amplification) were analyzed in 44 MDS patients treated Vidaza® (5-azacitidine, AC). Hypermethylation of at least one gene was detected in 9/44 patients (20.5%), including four genes CDKN2B, FHIT, ESR1 and IGSF4. Interestingly, of 9 hypermethylated patients, 8 patients showed demethylation in concordance with their clinical responses after three to five cycles AC treatment. Lack or decrease methylation was observed in four patients with hematological improvements. Persistence methylation was observed in four others who became AML transformation or no response after treatment, especially reinforcing methylated gene in a case progressed to leukemia later. Our study also founds out IGSF4 gene hypermethylation in MDS as a first report. Additionally, mRNA expression of CDKN2B, IGSF4, and ESR1 in MDS were significantly lower than those in the control group (p < 0.05). Our results suggest that the methylation changes of specific genes contributes to disease pathogenesis and might present a molecular marker that can be used to monitor the efficacy of AC treatment in MDS. Disclosures: No relevant conflicts of interest to declare.

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SATB1 Regulates Chromatin Organization and HSP70 Expression in Early Erythropoiesis and Is Downregulated in Models of Diamond Blackfan Anemia

Blood ◽

10.1182/blood-2021-153814 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2189-2189

Author(s):

Mark C Wilkes ◽

Aya Shibuya ◽

Vanessa M Scanlon ◽

Hee-Don Chae ◽

Anupama Narla ◽

...

Keyword(s):

Cord Blood ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Culture Media ◽

Hsp70 Expression ◽

Erythroid Progenitors ◽

Erythroid Progenitor ◽

Hematopoietic Stem ◽

Enhancer Element ◽

Diamond Blackfan Anemia

Abstract Diamond Blackfan Anemia (DBA) is a rare genetic disease predominantly caused by mutations carried within one of at least 20 ribosomal genes. DBA is characterized by red blood cell aplasia and normal myeloid and megakaryocyte progenitors, indicating that early uncommitted progenitors are relatively unaffected by the mutations. In DBA, the formation of BFU-E colonies and subsequent erythroblasts are severely restricted and indicate a defect in one of the earliest stages of erythroid expansion. To identify critical molecular mechanisms that may regulate early erythropoiesis, we used shRNAs against the ribosomal protein RPS19 (the most commonly mutated gene in DBA) in cord blood derived CD34+ hematopoietic stem and progenitor cells (HSPCs) and performed bulk RNA-seq. After 3 days in an erythroid culture media, the transcriptomes in CD71+ erythroid progenitors were examined. We found that the special AT binding protein 1 (SATB1) was downregulated in RPS19-insufficient HSPCs compared to healthy cord blood HSPCs. SATB1 is modestly expressed in hematopoietic stem cells but is induced during lymphoid expansion and has been previously reported to suppress myeloid/erythroid progenitor (MEP) expansion. Our results showed that maintaining SATB1 expression is required for optimal expansion of MEP progenitors and that the premature loss of SATB1 in DBA contributes to the anemia phenotype. SATB1 binds to 3 specific regions upstream of the 5'UTR of the HSP70 genes and induces the formation of 2 chromatin loops. An enhancer element associates with the proximal promoters of the two HSP70 genes and facilitates the induction of HSP70. In DBA, HSP70 is not induced and contributes to DBA pathogenesis. HSPA1A is induced 4.3-fold while HSPA1B is induced 3.1-fold. Increased expression of the master erythroid transcription factor GATA1 during erythropoiesis occurs in two phases. The first induction precedes a more dramatic induction that accompanies later stages of erythroid differentiation. The absence of SATB1 or HSP70 reduced the earlier GATA1 induction that accompany MEP expansion by 46.1% and 49.3% respectively. The number of MEPs in SATB1 knockdown HSPCs was reduced, resulting in a 24.5% reduction in CD235+ erythroid and 20.8% reduction in CD41+ megakaryocytes. While SATB1-independent effects of RPS19-insufficiency contribute more significantly to erythroid defects in DBA, we have uncovered that SATB1 contributes to regulation of the earliest stages of erythropoiesis by facilitating the induction of HSP70 and subsequent stabilization of an early induction of GATA1. Disclosures No relevant conflicts of interest to declare.

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The DNA Methylation Maintenance Protein UHRF1 Regulates Fetal Globin Expression Independent of HBG Promoter DNA Methylation

Blood ◽

10.1182/blood-2018-99-115589 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 410-410

Author(s):

Ruopeng Feng ◽

Phillip A Doerfler ◽

Yu Yao ◽

Xing Tang ◽

Yong-Dong Wang ◽

...

Keyword(s):

Dna Methylation ◽

Dna Methyltransferase ◽

Clinical Symptoms ◽

Conflicts Of Interest ◽

Globin Gene ◽

Gene Promoters ◽

Methylated Dna ◽

Cpg Sites ◽

Cd34 Cells ◽

Gene Switching

Abstract Pharmacological or genetic induction of fetal hemoglobin (HbF, α2γ2) in adult red blood cells is a proven strategy to ameliorate the clinical symptoms of sickle cell disease (SCD) and β-thalassemia. Therefore, efforts are underway to better understand mechanisms that mediate the perinatal switch from HbF to adult hemoglobin (HbA, α2β2). We performed a CRISPR-Cas9/guide (g) RNA screen to identify novel proteins that regulate HbF production in HUDEP-2 cells, a human erythroid line that normally expresses HbA. We identified UHRF1 (ubiquitin-like with PHD and RING finger domains 1) as a repressor of HbF production. UHRF1 binds hemi-methylated DNA and recruit DNA methyltransferase 1 (DNMT1) to ensure faithful maintenance of DNA methylation during DNA replication. Numerous UHRF1-interacting proteins, including DNMT1, EHMT1/2 and HDAC2 are associated with γ-globin repression. We used CRISPR/Cas9 and RNA interference to validate UHRF1 as a HbF regulator. Compared to non-targeting gRNA UHRF1 disruption using Cas9 + 2 separate gRNAs increased the γ-globin/γ+β-globin RNA ratio from 1.9 to 25.8/27.1% (P<0.01), increased the fraction of HbF immunostaining cells ("F-cells") from 7.5 to 25.1/35.4% and increased HbF protein from 2.10 to 16.3/15.0% (P<0.01) in HUDEP-2 cells. Compared to a control luciferase shRNA, 2 different UHRF1 shRNAs increased theγ-globin/γ+β-globin RNA ratio from 9.68% to 21.59/28.93% (P<0.01), increased the F-cell fraction from 37.9 to 49.8/55.6% and increased HbF protein from 9.1 to 16.18/18.5% (P<0.05) in erythroid cells derived from normal adult peripheral blood CD34+ cells. UHRF1 deficiency did not alter erythroid maturation or expression of key transcription factor genes that regulate HbF expression in HUDEP-2 or CD34+ cells (BCL11A, ZBTB7A, MYB and KLF1). UHRF1 mutant proteins defective in recognizing H3K9me2 (FW237/238AA), binding to hemi-methylated DNA (R491A) or ubiquitination of H3K23 to enhance DNMT1 recruitment (C741A), were unable to repress HBG1/HBG2. These mutations have the most profound effects on maintaining DNA methylation, indicating that UHRF1 represses HBG1/HBG2 in HUDEP-2 cells through this mechanism. UHRF1 knockout induced genome-wide demethylation including 6 CpG sites located at positions -162, -53, -50, +6, +17, +50 positions relative to the γ-globin (HBG1 and HBG2) transcription start site. Demethylation of these sites is thought to be required for γ-globin de-repression. However, forced demethylation of these cytosines in HUDEP-2 cells using specific gRNAs + dead (d) Cas9-TET1 was not sufficient to activate γ-globin expression when UHRF1 was present. Additionally, dCas9-DNMT3a-mediated methylation of the HBG promoter CpG residues in UHRF1 knockdown HUDEP-2 cells did not inhibit γ-globin expression in UHRF1 knockout HUDEP-2 cells. Based on these studies, we conclude that: 1) UHRF1 regulates γ-globin transcription; 2) demethylation of CpG sites at the HBG gene promoters is neither necessary or sufficient for γ-globin induction; 3) UHRF1 regulates γ-to-β globin gene switching either by methylating DNA regions other than those present around the HBG promoter or through non-canonical activities. Distinguishing these mechanisms will elucidate further our understanding of globin gene switching and could identify new pathways for pharmacological induction of HbF. Disclosures No relevant conflicts of interest to declare.

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Comparison of DNA Methyltransferase Inhibitors in Patients with Myelodysplastic Syndrome

Blood ◽

10.1182/blood.v124.21.5617.5617 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5617-5617

Author(s):

Esin Avsar ◽

Ozan Salim ◽

Tayfur Toptas ◽

Burak Deveci ◽

Erdal Kurtoglu ◽

...

Keyword(s):

Myelodysplastic Syndrome ◽

Dna Methyltransferase ◽

Transfusion Requirement ◽

Conflicts Of Interest ◽

Indirect Comparison ◽

Response Rates ◽

Refractory Anemia ◽

Hematologic Toxicity ◽

Dna Methyltransferase Inhibitors ◽

Significant Difference

Abstract The aim of this retrospective study was to compare the response rates (RR), overall survival (OS), time–to–acute myeloid leukemia–transformation (TTAMLT), hematologic toxicity and transfusion requirement of azacitidine and decitabine in patients with Myelodysplastic Syndrome Refractory Anemia with Excess Blasts (MDS RAEB) types 1 and 2. A total of 51 patients (35 male, 16 female, median age (years): 67) diagnosed with MDS RAEB-1 and RAEB-2 treated with azacitidine (n=31) or decitabine (n=20) in three Turkish institutes were evaluated . Response to treatment was classified according to International Working Group response criterias. Competing risk analysis was used to compare TTAMLT. There was no significant difference between the two groups in terms of age, gender, performance score, diagnosis, etiology, treatment cycles, bone marrow cellularity and blast percentage. Most patients received at least 4 cycles of azacitidine or decitabine. Patients who received azacitidine or decitabine had comparable response rates (58.1% vs 66.7%, respectively, p= 0.55). There was no difference in terms of red blood cell and platelet transfusion requirement and febrile neutropenia episodes (p= 0.46, p= 0.27, p= 0.19, respectively). Median follow–up was 31 months for azacitidine and 20 months for decitabine. Median OS was 18.4 months for azacitidine and 13.4 months for decitabine (p= 0.16). Only predictor of OS was at least 4-cycle treatment with any DNA methyltransferase inhibitors. 1–year risk of AML transformation was comparable across the groups (26% vs 33%, p= 0.55). In conclusion, both azacitidine and decitabine have similar efficacy and toxicity profiles in the treatment of MDS RAEB-1 and RAEB-2. Until a head–to–head comparison in prospective, randomized studies are conducted, the current source of available data will derive from meta–analyses that consist of the indirect comparison of treatment arms, or retrospective analyses with inevitable biases. Disclosures No relevant conflicts of interest to declare.

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Expression Of Mutant Spliceosomal Protein SF3B1 Results In Dysregulated Hematopoietic Maturation

Blood ◽

10.1182/blood.v122.21.2773.2773 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2773-2773

Author(s):

Alexander C. Minella ◽

Oscar Ramirez ◽

Yanfei Xu ◽

Tushar Murthy ◽

Xiaodong Yang ◽

...

Keyword(s):

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Erythroid Differentiation ◽

Retroviral Vectors ◽

Peptide Sequence ◽

Causative Role ◽

Wild Type ◽

Erythroid Maturation

Abstract Whole genome sequencing has recently revealed the prevalence of mutations in proteins directing splicing of RNA in up to half of the patients with Myelodysplastic Syndrome (MDS). Mutations in the protein SF3B1 are particularly common in MDS patients with the phenotypic abnormality termed ring sideroblasts (dysplastic erythroid precursors with perinculear rings formed by iron-laden mitochondria). The most common SF3B1 mutation in MDS patients results in a change from lysine to glutamic acid at amino acid position 700 (K700E). Given that splicing of RNA is a ubiquitous phenomenon, it is unclear how these mutations result in clonal proliferation and dysplastic hematopoiesis; two hallmark features of MDS. Furthermore, direct experimental evidence demonstrating a causative role for SF3B1 mutations in MDS-related phenotypes is lacking. To better understand how mutations of spliceosomal proteins contribute to MDS pathogenesis, we sought to define how expression of mutant SF3B1 changes erythroid maturation in vitro and in vivo. Native SF3B1 cDNA constructs are not amenable to bacterial propagation due to toxicity of its HEAT-domain repeats. We overcame this problem by codon optimization (changing the DNA sequence while preserving the native peptide sequence). Human cord blood derived CD34+ cells were transduced with retroviral vectors to express either the wild-type or K700E mutant of SF3B1. After a week of expansion in cytokines (IL-3, SCF and IL6), cells were induced to erythroid differentiation by addition of erythropoietin (EPO) and analyzed for surface markers of erythroid differentiation (CD 71, CD117, CD105, CD45 and CD235A) at regular intervals. K700E mutant expressing cells were found to have significantly reduced expression of CD105 when compared to wild-type SF3B1-expressing cells (average 50% recuction, n =8). CD105 or endoglin is a TGF-beta receptor accessory receptor expressed at high levels during intermediate stages of erythroid maturation. A more modest reduction of CD71 expression was also noted in K700E-SF3B1 cells. MDS bone marrow is known to express low levels of both CD105 and CD71 making our results clinically relevant. To further characterize how mutant SF3B1 may cause dysplastic hematopoiesis, we studied transduced and transplanted murine progenitor cells in vivo and in colony forming assays. Murine data demonstrate significantly reduced K700E-transduced hematopoietic progenitors (as defined by flow-cytometry) in vivo and impaired erythroid colony formation in vitro. Together, our results suggest that enforced expression of K700E-SF3B1 induces aberrant erythroid maturation and impairs homeostasis of hematopoietic precursor cells. Thus, we provide direct evidence that MDS-associated SF3B1 mutations perturb normal hematopoiesis and offer rationale for using our complementary experimental approach as a platform for elucidating the molecular mechanisms through which mutations in RNA splicing factors promote hematologic disease. Disclosures: No relevant conflicts of interest to declare.

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Stable DNMT3L Overexpression in SH-SY5Y Neurons Recreates a Facet of the Genome-Wide Down Syndrome DNA Methylation Signature

10.1101/2020.11.09.374793 ◽

2020 ◽

Author(s):

Benjamin I. Laufer ◽

J. Antonio Gomez ◽

Julia M. Jianu ◽

Janine M. LaSalle

Keyword(s):

Dna Methylation ◽

Down Syndrome ◽

Dna Methyltransferase ◽

Molecular Mechanisms ◽

Cpg Island ◽

Neuroblastoma Cell ◽

Chromosome 21 ◽

Pairwise Comparisons ◽

Genome Wide ◽

A Genome

AbstractDown syndrome (DS) is characterized by a genome-wide profile of differential DNA methylation that is skewed towards hypermethylation in most tissues, including brain. The molecular mechanisms involve the overexpression of genes related to DNA methylation on chromosome 21. Here, we stably overexpressed the chromosome 21 gene DNA methyltransferase 3L (DNMT3L) in the human SH-SY5Y neuroblastoma cell line and assayed DNA methylation at over 26 million CpGs by whole genome bisulfite sequencing at three different developmental phases (undifferentiated, differentiating, and differentiated). DNMT3L overexpression resulted in global CpG and CpG island hypermethylation as well as thousands of differentially methylated regions (DMRs). The DNMT3L DMRs were skewed towards hypermethylation and mapped to genes involved in neurodevelopment, cellular signaling, and gene regulation. Merging the DMRs into a consensus profile where the cell lines clustered by genotype and then phase demonstrated that different regions of common genes are affected. The hypermethylated DMRs from all pairwise comparisons were enriched for regions of bivalent chromatin marked by H3K4me3 as well as differentially methylated CpGs from previous DS studies of diverse tissues. In contrast, the hypomethylated DMRs from all pairwise comparisons displayed a tissue-specific profile enriched for regions of heterochromatin marked by H3K9me3 during embryonic development. Taken together, we propose a mechanism whereby regions of bivalent chromatin that lose H3K4me3 during development are targeted by excess DNMT3L and become hypermethylated, while excess DNMT3L also evicts DNMT3A from heterochromatin, resulting in hypomethylation. Overall, these findings demonstrate that DNMT3L overexpression during neurodevelopment recreates a facet of the DS DNA methylation signature.

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