Histone demethylase Lsd1 represses hematopoietic stem and progenitor cell signatures during blood cell maturation
- Boston Children's Hospital and Dana-Farber Cancer Institute, Harvard Medical School, United States
- Howard Hughes Medical Institute, Harvard Medical School, United States
Figures
Figure 1 with 1 supplement
Deletion of Lsd1 results in pancytopenia.
(A) Lsd1fl/fl VavCre cohort statistics 0 and 10 days after birth (n = 146 pups). (B) Lsd1fl/fl and Lsd1fl/fl VavCre newborn pups. (C) Western blot analysis of E13.5 fetal liver and newborn bone …
Figure 1—figure supplement 1
Lsd1 targeting strategy.
(A) Illustration of Lsd1 protein domain structure. (B) Mouse Lsd1 locus (Chr4. D3) drawn to scale; Untargeted wild-type allele (exons 3–8); targeted allele +neo (fl_neo/+): wild-type exons 5 and 6 …
Figure 2 with 2 supplements
Lsd1 knockout HSCs fail to give rise to terminally differentiated cells.
(A) Immunophenotypic analysis of control and Lsd1fl/fl Mx1Cre hematopoietic stem and progenitor cells 1 week after final poly(I:C) dose. (B) Alternative gating logic for analysis of Lsd1fl/fl Mx1Cre …
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Figure 2—source data 1
Source data for Figure 2—figure supplement 2. Background corrected CT values of FACS-sorted single cells analyzed by BIOMARK Fluidigm microfluidics qPCR.
- https://doi.org/10.7554/eLife.00633.006
Figure 2—figure supplement 1
Mx1Cre-mediated deletion of Lsd1 results in pancytopenia.
(A) Experimental design. (B) Western Blot analysis of three control (Lsd1fl/fl) and three experimental (Lsd1fl/fl Mx1Cre) spleen lysates 1 week after p(I:C) injection. (C) Genomic PCR from control …
Figure 2—figure supplement 2
Hierarchical clustering of immunoprofiles derived from single sorted Lsd1 knockout CD150+ CD48− LS+K+ and LS+K− cells.
(A) Frequency of Lsd1fl/fl and Lsd1fl/fl Mx1Cre CD150+ CD48− LS+K+ LT-HSCs in G1/G0 vs S/G2/M cell cycle phases. Unfractionated bone marrow cells were incubated with Hoechst 33342, stained with …
Figure 3
Lsd1 is essential for HSC self-renewal.
(A) Experimental outline for competitive transplantation experiment. Equal numbers of unfractionated CD45.2 competitor bone marrow cells were mixed with equal numbers of either CD45.1/.2 Lsd1fl/fl …
Figure 4 with 1 supplement
Lsd1 is required for terminal granulocytic and erythroid differentiation.
(A) Flow cytometric analysis for indicated myeloid cell surface markers 1 week after poly(I:C). (B) Quantification and statistical analysis of Gr1/Mac1 and Neutr.7/4/F4/80 flow cytometric analysis. …
Figure 4—figure supplement 1
Lsd1fl/fl EpoRCre mice die in utero due to erythroid differentiation defects.
(A) Frequency of early (Annexin V+ 7-AAD−) and late (Annexin V+ 7-AAD+) apoptotic Lsd1fl/fl and Lsd1fl/fl Mx1Cre Gr1dim Mac1+ bone marrow cells (left), as well as, early (Annexin V+ 7-AAD−) and late …
Figure 5 with 1 supplement
Loss of Lsd1 results in derepression of stem and progenitor cell genes.
(A)–(C) Gene set enrichment analyses showing upregulation of LSK signature genes in Lsd1fl/fl Mx1Cre Gr1dim Mac1+ myeloid cells (A), Lsd1fl/fl EpoRCre CD71+ c-Kit+ erythroid cells (B), and Lsd1fl/fl …
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Figure 5—source data 1
Gene sets used for GSEA analyses.
- https://doi.org/10.7554/eLife.00633.013
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Figure 5—source data 2
Normalized enrichment scores (NES) and false discovery rates (FDR) of GSEA analyses with stem/progenitor gene sets.
- https://doi.org/10.7554/eLife.00633.014
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Figure 5—source data 3
Lsd1 occupancy peak position in 32D cells.
- https://doi.org/10.7554/eLife.00633.015
Figure 5—figure supplement 1
Deletion of Lsd1 results in upregulation of stem and progenitor cell genes.
(A) Heatmap of normalized log2 expression values of control and Lsd1fl/fl Mx1Cre Gr1dim Mac1+ cells (twofold or less changes; p≤0.05). (B) Relative mRNA expression levels of myeloid and erythroid …
Figure 6 with 2 supplements
Lsd1 occupies enhancers and promoters of stem and progenitor cell genes and loss of Lsd1 is associated with increased levels of H3K4me1 and H3K4me2 on enhancers and promoters of HSPC genes.
(A) Venn diagram of the overlap between Lsd1 and promoter-associated H3K4me2 and H3K4me3 peaks in wild-type Gr1dim Mac1+ cells. (B) Venn diagram of overlap between Lsd1 with enhancer-associated …
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Figure 6—source data 1
Target genes of Lsd1 KO-specific and wt-specific histone modification peaks in Gr1dim Mac1+ cells.
- https://doi.org/10.7554/eLife.00633.018
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Figure 6—source data 2
Target genes of Lsd1 in 32D cells (distal; promoter; distal, and promoter).
- https://doi.org/10.7554/eLife.00633.019
Figure 6—figure supplement 1
Validation of Lsd1 ChIP-Seq and normalization of histone ChIP-Seq data using ‘MAnorm’.
(A) In vivo chromatin occupancy was examined by ChIP-qPCR in primary murine Gr1dim Mac1+ myeloid cells. ChIP-qPCR validation for Lsd1 binding on LSK genes compared to rabbit IgG and negative control …
Figure 6—figure supplement 2
Definition of Lsd1 knockout unique histone methylation peaks.
(A)–(D) Histone modification peaks (i.e., H3K4me1, H3K4me2, H3K4me3, and H3K27ac) derived from Lsd1fl/fl (wild type) and Lsd1fl/fl Mx1Cre (Lsd1 KO) Gr1dim Mac1+ cell ChIP-Seq experiments were …
Figure 7
Cellular and molecular effects of Lsd1 loss during hematopoietic differentiation.
(A) Characterization of the stem/progenitor cell compartment of Lsd1 knockout mice demonstrated the presence LT-HSCs (CD150+ CD48− LSK), while having lost all LS−K+ myeloid progenitor cells (i.e., …
