Journal: Oncotarget

Article Title: Factors involved in CLL pathogenesis and cell survival are disrupted by differentiation of CLL B-cells into antibody-secreting cells

doi:

Figure Lengend Snippet: A. The expression of the LEF1 , TCL1 , ROR1 , FMOD , TNFRSF13B , ATXN1 , MARCKS , BIRC5 , FCER2 , Ly9 and EBF1 genes was evaluated by quantitative real-time RT-PCR on D0 and D7. Results are expressed relative to gene expression in CLL B-cells on D0, according to the 2 −ΔΔCT method. The results are represented as log2 fold changes in box-and-whisker (min to max) plots (the “+” sign indicates the mean) for 11 experiments. Statistical significance was calculated using the Wilcoxon's test: * p < 0.05, ** p < 0.01, ns, not significant. B. Immunoblot analysis and densitometry quantification of IRF4, BLIMP1, LEF1 full length and ΔN LEF-1 isoforms, ROR1, p27, FMOD, survivin, CD229, PI3K and BTK in cells from three CLL samples at D0 and D7. Ramos, RPMI8226 and LP1 cell lines were used as controls. Statistical significance was calculated using Student's t -test: * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: [ ] The TaqMan Gene Expression assays for PRDM1 (BLIMP1) (assay ID Hs00153357_m1), IRF4 (Hs01056533_m1), XBP1s (Hs03929085_g1), PAX5 (Hs00172003_m1), BCL6 (Hs00277037_m1), IRF8 (Hs01128710_m1), BACH2 (Hs00222364_m1), BATF (Hs00232390_m1), GAS6 (Hs01090305_m1), CD138 (Hs00896423_m1), LEF1 (Hs01547250_m1), TCL1A (Hs00951350_m1), ROR1 (Hs00938677_m1), FCER2 (Hs00233627_m1), BIRC5 (HS04194392_s1), FMOD (Hs00157619_m1), MARCKS (Hs00158993_m1), ATXN1 (Hs00165656_m1), Ly9 (Hs03004330_m1), TNFRSF13B (Hs00963364_m1) and EBF1 (Hs00395524_m1) were purchased from Applied Biosystems.

Techniques: Expressing, Quantitative RT-PCR, Gene Expression, Whisker Assay, Western Blot

Journal: American Journal of Physiology - Cell Physiology

Article Title: The impact of skeletal muscle contraction on CD146 + Lin − pericytes

doi: 10.1152/ajpcell.00156.2019

Figure Lengend Snippet: Primer information and gene expression assay ID numbers

Article Snippet: Primer information and gene expression assay ID numbers used in this study are provided in . table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Gene Symbol Gene Name Assay ID Ang Angiogenin Mm00833184_s1 Bdnf Brain-derived neurotrophic factor Mm00833184_s1 Col1a1 Collagen type I α 1 chain Mm00801666_g1 Col3a1 Collagen type III α1 chain Mm01254476_m1 Col6a3 Collagen type VI α3 chain Mm00711678_m1 Ebf1 Early B cell factor 1 Mm00432954_m1 Egf Epidermal growth factor Mm00438696_m1 Fgf2 Fibroblast growth factor 2 Mm01285715_m1 Fndc5 Fibronectin type III domain containing 5 Mm01181543_m1 Hgf Hepatocyte growth factor Mm01135184_m1 Igf1 Insulin like growth factor 1 Mm00439560_m1 Igf2 Insulin like growth factor 2 Mm00439564_m1 Lama2 Laminin subunit α 2 Mm00550083_m1 Lif Leukemia inhibitory factor Mm00434762_g1 Mmp2 Matrix metallopeptidase 2 Mm00439498_m1 Mmp9 Matrix metallopeptidase 9 Mm00442991_m1 Mmp14 Matrix metallopeptidase 14 Mm00485054_m1 Myf5 Myogenic factor 5 Mm00435125_m1 Myod1 Myogenic differentiation 1 Mm00440387_m1 Myog Myogenin Mm00446194_m1 Ngf Nerve growth factor Mm00443039_m1 Ntf3 Neurotrophin 3 Mm00435413_s1 Pax3 Paired box 3 Mm00435491_m1 Pax7 Paired box 7 Mm01354484_m1 Pparg Peroxisome proliferator activated receptor-γ Mm00440940_m1 Tcf4 Transcription factor 4 Mm00443210_m1 Timp1 TIMP metallopeptidase inhibitor 1 Mm01341361_m1 Timp2 TIMP metallopeptidase inhibitor 2 Mm00441825_m1 Vegfa Vascular endothelial growth factor A Mm00437306_m1 Zfp423 Zinc finger protein 423 Mm00677660_m1 Open in a separate window Primer information and gene expression assay ID numbers RNA sequencing RNA was extracted from cell lysates using miRNeasy Mini Kit (Qiagen), following the manufacturer’s instruction (RNAs ≥18 nt).

Techniques: Gene Expression

Journal: bioRxiv

Article Title: Single-nucleus transcriptomics reveals functional compartmentalization in syncytial skeletal muscle cells

doi: 10.1101/2020.04.14.041665

Figure Lengend Snippet: a , Marker genes enriched in MTJ-A or MTJ-B nuclei are presented by violin plots. b , Upper two rows – single molecule FISH of two MTJ-A markers ( Tigd4 and Col22a1 ) in uninjured or 14 d.p.i TA muscle expressing H2B-GFP in myonuclei. Bottom row – Ebf1 (MTJ-B marker) immunoflourescence in uninjured TA muscle expressing H2B-GFP in myonuclei. Shown are MTJ regions; T, tendon and M, myofiber. Arrowheads indicate co-localization of MTJ marker genes and GFP. Scale bar, 30µm. c , Conventional FISH experiment in an isolated single EDL fiber. Insets show magnification of MTJ (i) and NMJ (ii) regions. Scale bars, 100 µm (for the entire fiber) and 30 µm (for the insets). Expression patterns were validated in 2 or more individuals.

Article Snippet: The antibodies used for this study were β-actin (Cell Signaling, 1:1000), ColVI (Abcam, 1:2000) and Ebf1 (1:1000).

Techniques: Marker, Expressing, Isolation

Journal: bioRxiv

Article Title: Single-nucleus transcriptomics reveals functional compartmentalization in syncytial skeletal muscle cells

doi: 10.1101/2020.04.14.041665

Figure Lengend Snippet: a , Heat-map showing the expression level of transcription factors and epigenetic regulators in indicated nuclear subtypes; see also Supplementary Table 3 for the full list including nuclear subtypes not shown in this Figure. b , Ebf1 directly binds to genomic regions of the top 100 genes identified to be specifically expressed in MTJ-B nuclei (ENCODE project - accession number ENCSR000DZQ), but much less to markers of MTJ-A or NMJ. Classification of the Ebf1 binding sites in these 100 genes. c , Western blot analysis of C2C12 cell line expressing doxycycline-inducible Ebf1. d , Indicated genes were analyzed by RT-qPCR before/after inducing Ebf1 expression. Dox treatment in control cells (no Ebf1) did not have any effect (data not shown). Error bars indicate S.E.M. Two tailed paired student’s t-test between untreated and Dox treated cells (n=3). *, p < 0.05. **, p < 0.01. ***, p < 0.001. e , Single molecule FISH of indicated marker genes in TA muscle of control or Ebf1 mutant mice. Arrows indicate myonuclei expressing MTJ-B marker genes. T, tendon. Downregulation of expression was validated in 4 individuals. Scale bar, 50 µm.

Article Snippet: The antibodies used for this study were β-actin (Cell Signaling, 1:1000), ColVI (Abcam, 1:2000) and Ebf1 (1:1000).

Techniques: Expressing, Binding Assay, Western Blot, Quantitative RT-PCR, Control, Two Tailed Test, Marker, Mutagenesis

Journal: The Journal of Experimental Medicine

Article Title: EBF1 primes B-lymphoid enhancers and limits the myeloid bias in murine multipotent progenitors

doi: 10.1084/jem.20212437

Figure Lengend Snippet: EBF1 expression in MPP3 and MPP4 populations are required for normal HSPC composition. (A) Normalized read counts of Ebf1 in different hematopoietic cell populations from public RNA-seq datasets (original data from [left] and GEO dataset GSE162662 [right]). Data from GEO dataset GSE162662 representing CLP and GMP populations are represented on a different axis scale than data from representing LT-HSC, MPP1, MPP2, MPP3, and MPP4 populations. (B) qRT-PCR of Ebf1 mRNA expression relative to Actb , normalized to WT Ebf1 CLP (Lin − Sca1 int cKit int IL7R + Flt3 + ) expression. Data are represented as mean ± SEM. n = 3–10. (C) Relative expression of Ebf1 and Ash2l by RT-PCR. The first outer PCR was performed with 10 cycles and the second PCR was performed with increasing cycle numbers (25, 30, 35, and 40 cycles). The product of the second PCR was visualized on an agarose gel. Image is representative of three experiments. (B and C) cDNA was generated from 10,000 FACS sorted cells of the indicated populations. (D) Immunoblot analysis of EBF1 protein levels in total cell extracts of 150,000 FACS sorted cells of HSC (LSK CD150 + CD48 − ), MPP2 (LSK CD150 + CD48 + ), MPP3 (LSK CD150 − CD48 + ), MPP4 (LSK CD150 − CD48 + Flt3 + ), CLPs (Lin − cKit int Sca1 int IL7R + Flt3 + ), splenic B (CD19 + ) cells, and Fr. A (pre-pro-B) cells from WT Ebf1 mice. T (CD4 + CD8 + ) cells and Ebf1 KO Fr. A cells were used as a negative control. Histone H3 was used as a loading control. Immunoblot is representative of three experiments. (E) Absolute number of CD19 + B cells in the BM. Ebf1 WT and Ebf1 KO n = 7, Ighm WT and Ighm Tm1 n = 11. (F) Total BM cellularity. Ebf1 WT n = 24, Ebf1 KO n = 14, Ighm WT and Ighm Tm1 n = 11. (G) Boxplots showing the absolute number of HSC, erythroid-platelet biased MPP2, myeloid-biased MPP3, and lymphoid-biased MPP4 populations in the BM. Ebf1 WT n = 19–23, Ebf1 KO n = 14–18, Ighm WT and Ighm Tm1 n = 8. (H) Absolute number of CLP cells in the BM. (I) Proportion of ALP and BLP cells in the CLP population. (H and I) Ebf1 WT n = 10, Ebf1 KO n = 4, Ighm WT and Ighm Tm1 n = 4. (J) Schematic of reverse AdT assays with Ebf1 WT and Ebf1 KO mice. 5 × 10 6 total BM cells of WT mice (CD45.1) were injected into lethally irradiated Ebf1 WT and Ebf1 KO recipients (CD45.2). (K) Boxplots showing the frequency of CD45.1 donor cells in the BM of Ebf1 WT and Ebf1 KO recipients, 16 wk after AdT. (L) Boxplots showing the frequencies of HSCs, MPP2, MPP3, and MPP4 within CD45.1 donor cells in the BM of the Ebf1 WT and Ebf1 KO recipients 16 wk after AdT. (K and L) Ebf1 WT n = 13, Ebf1 KO n = 14. (E–L) Statistical significance was determined by Mann–Whitney U test. (B–L) Data are from >2 independent experiments. Source data are available for this figure: .

Article Snippet: PCR products were resolved on 2% agarose gels. qRT-PCR was performed with Taqman Gene Expression Assays (FAM; Thermo Fisher Scientific) with Taqman Fast Advanced Master Mix (Thermo Fisher Scientific) using target probes against Ebf1 (Mm01288947_g1) or Cebpa (Mm01265914_s1) normalized to Act2b (Mm00607939_s1 Actb).

Techniques: Expressing, RNA Sequencing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Generated, Western Blot, Negative Control, Control, Injection, Irradiation, MANN-WHITNEY

Journal: The Journal of Experimental Medicine

Article Title: EBF1 primes B-lymphoid enhancers and limits the myeloid bias in murine multipotent progenitors

doi: 10.1084/jem.20212437

Figure Lengend Snippet: Extended characterization of HSC functionality upon Ebf1 deficiency. Related to and . (A) Gating strategies for HSC, MPP2, MPP3, and MPP4 cells. (B) Gating strategies for CLP, ALP, and BLP cells. Representative plots are shown for Ebf1 WT and Ebf1 KO mice. (C) Frequencies of myeloid cells (CD11b + ), B cells (CD19 + ), and T cells (CD4 + CD8 + ) within CD45.1 donor cells in the BM of the Ebf1 WT and Ebf1 KO recipients 16 wk after AdT. Ebf1 WT n = 13, Ebf1 KO n = 14. (D) Absolute number of CD41 + HSCs in the BM. Ebf1 WT n = 11, Ebf1 KO n = 9, Ighm WT n = 11, and Ighm Tm1 n = 14. (E) 300 WT CTY-labeled HSCs were cultured in TPO and SCF, with increased concentrations of BM fluid collected from Ebf1 WT and Ebf1 KO mice. Division index (CTY mean fluorescence intensity [MFI] from day n/CTY MFI from day 0) at day 2 and day 5, of CTY-labeled HSCs cultured with different BM fluid concentrations from Ebf1 WT and Ebf1 KO mice, n = 6–8. (F) CFU assay performed with 150 sorted HSCs plated in triplicate in methocult. Total number of colonies formed after 10–12 d of plating. Biological replicates Ebf1 WT and Ebf1 KO n = 2, Ighm WT and Ighm Tm1 n = 3. (G) CFU assay performed with 150 sorted LT-HSCs (LSK CD34 − Flt3 − CD150 + CD48 − ) plated in triplicate in methocult. Colony identification after 10–12 d of plating. Biological replicates Ebf1 WT and Ebf1 KO n = 5. (H) Frequency of CD45.2 donor-derived cells in the peripheral blood within T cells (CD4 + CD8 + ; ****, P < 0.0001; ***, P = 0.0004; **, P = 0.0013), myeloid cells (CD11b + ; ***, P = 0.0002; ****, P < 0.0001; **, P = 0.0010; **, P = 0.0024) and B cells (CD19 + ; **, P = 0.0060; ****, P < 0.0001), during the AdT. Data are represented as mean ± SEM. Ebf1 WT n = 33 and Ebf1 KO n = 40, Ighm WT n = 17, and Ighm Tm1 n = 19. (C–H) Statistical significance was determined by Mann-Whitney U test. Data are from >2 independent experiments.

Article Snippet: PCR products were resolved on 2% agarose gels. qRT-PCR was performed with Taqman Gene Expression Assays (FAM; Thermo Fisher Scientific) with Taqman Fast Advanced Master Mix (Thermo Fisher Scientific) using target probes against Ebf1 (Mm01288947_g1) or Cebpa (Mm01265914_s1) normalized to Act2b (Mm00607939_s1 Actb).

Techniques: Labeling, Cell Culture, Fluorescence, Colony-forming Unit Assay, Derivative Assay, MANN-WHITNEY

Journal: The Journal of Experimental Medicine

Article Title: EBF1 primes B-lymphoid enhancers and limits the myeloid bias in murine multipotent progenitors

doi: 10.1084/jem.20212437

Figure Lengend Snippet: Reduced HSC quiescence and self-renewal capacity upon Ebf1 deletion. (A) Representative contour plots showing CD41 + expression on HSCs in Ebf1 WT and Ebf1 KO mice. (B) Boxplots showing the percentage of CD41 + expressing cells in the HSC population. Ebf1 WT n = 11, Ebf1 KO n = 9, Ighm WT and Ighm Tm1 n = 11. (C) Percentage of HSCs within each cell cycle phase (G0, G1, G2/S/M). Ebf1 WT n = 16, Ebf1 KO n = 16, Ighm WT and Ighm Tm1 n = 11. Data are represented as mean ± SEM. (D) Kaplan–Meier survival curve of WT, Ighm Tm1 , and Ebf1 KO mice following 5-FU injections every 10 d. Ebf1 WT and Ighm WT mice are represented together as WT mice. Ebf1 WT n = 14, Ebf1 KO n = 12, Ighm WT n = 10 and Ighm Tm1 n = 12. Statistical analysis was performed using the log-rank (Mantel-Cox) test. (E) Schematic showing primary AdT assays with Ebf1 WT , Ebf1 KO , Ighm WT , and Ighm Tm1 mice and secondary AdT assays with Ebf1 WT and Ebf1 KO mice. Lethally irradiated primary WT recipients (CD45.1.2) were injected with 150 HSCs from the indicated donors (CD45.2), together with 5 × 10 5 supportive (CD45.1) BM cells. Lethally irradiated secondary WT recipients (CD45.1.2) were injected with 3 × 10 6 total BM cells from the indicated primary recipient donors. (F) Boxplots showing frequency of CD45.2 donor cells in the BM of primary recipients, 16 wk after AdT. (G) Boxplots showing the frequencies of donor-derived cells within HSCs, T cells (CD4 + CD8 + ), B cells (CD19 + ), and myeloid cells (CD11b + ) in the BM of the primary recipients 16 wk after AdT. (F and G) Ebf1 WT n = 23, Ebf1 KO n = 25, Ighm WT n = 23 and Ighm Tm1 n = 26. (H) Boxplots showing frequency of CD45.2 donor cells in the BM of secondary recipients, 16 wk after AdT. (I) Boxplots showing the frequencies of donor-derived cells within HSCs, T cells, B cells, and myeloid cells in the BM of the secondary recipients 16 wk after AdT. (H and I) Ebf1 WT n = 23, Ebf1 KO n = 20. (J) 50,000 CTY-labeled LSK cells from Ebf1 WT and Ebf1 KO mice (CD45.2) were injected into lethally irradiated WT recipients (CD45.1). Frequency of CTY positive cells in the BM 14 h after AdT, fold change relative to Ebf1 WT . Ebf1 WT n = 6, Ebf1 KO n = 5. (B, C, and F–J) Statistical significance was determined by Mann–Whitney U test. (A–J) Data are from >2 independent experiments. HSCs defined as LSK CD150 + CD48 − .

Article Snippet: PCR products were resolved on 2% agarose gels. qRT-PCR was performed with Taqman Gene Expression Assays (FAM; Thermo Fisher Scientific) with Taqman Fast Advanced Master Mix (Thermo Fisher Scientific) using target probes against Ebf1 (Mm01288947_g1) or Cebpa (Mm01265914_s1) normalized to Act2b (Mm00607939_s1 Actb).

Techniques: Expressing, Irradiation, Injection, Derivative Assay, Labeling, MANN-WHITNEY

Journal: The Journal of Experimental Medicine

Article Title: EBF1 primes B-lymphoid enhancers and limits the myeloid bias in murine multipotent progenitors

doi: 10.1084/jem.20212437

Figure Lengend Snippet: Chromatin accessibility changes in Ebf1-deficient MPP3 and MPP4 cells. Related to . (A) Comparison of chromatin accessibility changes to gene expression changes in MPP3 versus MPP4 cells, in Ebf1 WT conditions. The y axis represents log 2 fold changes of gene expression, and the x axis represents log 2 fold changes of peak read counts in MPP3 versus MPP4 cells in Ebf1 WT conditions. Left: Dots represent ATAC peaks that overlap their TSS (TSS peaks). Right: Dots represent DA peaks between MPP3 versus MPP4 cells in Ebf1 WT conditions. Biological replicates for RNA-seq n = 4. Biological replicates for ATAC-seq n = 2. (B) Sequence logos of top ranked enriched motifs underlying the DA peaks in MPP3 and MPP4 cells from Ebf1 WT and Ebf1 KO mice. P values are depicted in red, percentages in blue reflect the fraction of hits found in the peak set, percentages in black reflect the fraction of hits found in the background set. (C) Immunoblot analysis of EBF1 protein levels in total cell extracts of 150,000 FACS-sorted cells of lineage-negative GFP + cells transduced with empty vector or EBF1 vector, biological replicates for ATAC-seq used in ( n = 4). Ebf1 WT MPP3 cells and CLPs (Lin − cKit int Sca1 int IL7R + Flt3 + ) were used a positive control. T (CD4 + CD8 + ) cells and Ebf1 KO Fr. A cells were used as a negative control. Histone H3 was used as a loading control. (D) Heatmaps of co-occurrence counts in KO Gained MPP3 peaks and KO Reduced and KO Gained MPP4 peaks of enriched motifs in these DA peak sets versus co-occurrence counts in all MPP3 and MPP4 peaks (1,000 replicates), respectively. Co-occurrence counts with z -scores >5 are displayed. (E) Enrichment analysis of genes associated with KO Reduced MPP4 peaks. (F) Genome tracks showing ATAC signal and EBF1 ChIP signal at B-lymphoid related KO Reduced MPP3 peaks annotated to the Cd19 gene. Original data from ; . Source data are available for this figure: .

Article Snippet: PCR products were resolved on 2% agarose gels. qRT-PCR was performed with Taqman Gene Expression Assays (FAM; Thermo Fisher Scientific) with Taqman Fast Advanced Master Mix (Thermo Fisher Scientific) using target probes against Ebf1 (Mm01288947_g1) or Cebpa (Mm01265914_s1) normalized to Act2b (Mm00607939_s1 Actb).

Techniques: Comparison, Gene Expression, RNA Sequencing, Sequencing, Western Blot, Transduction, Plasmid Preparation, Positive Control, Negative Control, Control

Journal: The Journal of Experimental Medicine

Article Title: EBF1 primes B-lymphoid enhancers and limits the myeloid bias in murine multipotent progenitors

doi: 10.1084/jem.20212437

Figure Lengend Snippet: EBF1 primes the B-lymphoid fate in myeloid-biased MPP3 progenitors. (A and B) Heatmap displaying chromatin accessibility at DA peaks in MPP3 and MPP4 cells (A) and in LT-HSCs (LSK CD34 − Flt3 − CD150 + CD48 − ; B) from Ebf1 WT and Ebf1 KO mice. Biological replicates n = 2. (A, B, and D) Peaks are organized into KO Reduced and KO Gained peaks, in MPP3 cells and MPP4 cells. Regions ± 3 kb around the center of the peak are shown. Heatmap scale represents RPKM. (C) Aggregation plots showing Tn5 activity in MPP3 and MPP4 cells from Ebf1 WT and Ebf1 KO conditions, at KO Reduced MPP3 peaks centered around the EBF1 motif (top row). Median aggregation plot for 100 random sets of CCCTC-binding factor (CTCF) motifs found in the ATAC peak set (bottom row). The shaded area depicts the SD. (D) Heatmap displaying chromatin accessibility at DA peaks in MPP3 cells from Ebf1 KO mice upon EBF1 re-expression and empty vector expression. (E) Heatmap displaying z-scores of co-occurrence counts in KO Reduced MPP3 peaks versus co-occurrence counts in all MPP3 peaks (1,000 replicates) of enriched motifs in KO Reduced MPP3 peaks. (F) Heatmap displaying average ChIP signal for EBF1 and PU.1 ChIPs at DA peaks. The RPKM signal is scaled over all four DA peak sets. (G) Enrichment analysis of genes associated with KO Reduced MPP3 peaks. (H) Genome tracks showing ATAC signal and EBF1 ChIP signal at B-lymphoid related KO Reduced MPP3 peaks annotated to the Igll1 gene. (I) Absolute number of CD19 + B cells at indicated time points upon EBF1 re-expression and empty vector expression, in Ebf1 WT and Ebf1 KO MPP3 and MPP4 cells. Data are from >2 independent experiments. (F and H) Original data from ; .

Article Snippet: PCR products were resolved on 2% agarose gels. qRT-PCR was performed with Taqman Gene Expression Assays (FAM; Thermo Fisher Scientific) with Taqman Fast Advanced Master Mix (Thermo Fisher Scientific) using target probes against Ebf1 (Mm01288947_g1) or Cebpa (Mm01265914_s1) normalized to Act2b (Mm00607939_s1 Actb).

Techniques: Activity Assay, Binding Assay, Expressing, Plasmid Preparation

Journal: The Journal of Experimental Medicine

Article Title: EBF1 primes B-lymphoid enhancers and limits the myeloid bias in murine multipotent progenitors

doi: 10.1084/jem.20212437

Figure Lengend Snippet: Role of Ebf1 deficiency in myeloid-biased transcriptome. Related to and . (A) PCA of bulk RNA-seq data from MPP3 and MPP4 cells of Ebf1 WT and Ebf1 KO conditions; based on DE genes. n = 4 biological replicates. (B) MA plots of MPP3 and MPP4 cells comparing Ebf1 WT and Ebf1 KO transcriptomes. Dots in blue represent genes with an FDR <0.1. The y axis represents shrunken log 2 fold change (FC), capped at |log 2 FC| 2, and the x axis represents log 10 basemean. (C and D) Comparison of chromatin accessibility changes to gene expression changes in MPP3 (C) and MPP4 (D) cells upon Ebf1 deletion. The y axis represents shrunken log 2 fold changes of gene expression and the x axis represents log 2 fold changes of peak read counts in Ebf1 WT versus Ebf1 KO MPP4 cells. Left: Dots represent DA peaks in MPP3 (C) and MPP4 (D) cells. Right: Dots represent DE genes and ATAC peaks that overlap their TSS (TSS peaks). (E) CFU assay performed with 150 sorted MPP3 and MPP4 cells plated in triplicate in methocult. Total number of colonies formed after 10–12 d of plating. Biological replicates Ebf1 WT and Ebf1 KO n = 6. Statistical significance was determined by unpaired t test. (F) Heatmap showing gene expression of DE genes in MPP3 and MPP4 cells, between Ebf1 WT and Ebf1 KO conditions. Genes are organised into MPP3-specific, common, and MPP4-specific DE genes. Genes with an annotated C/EBPα ChIP peak (original data from ; ) are labeled in blue. Heatmap scale represents z -scores calculated separately for MPP3 and MPP4 cells. (G) qPCR analysis of Cebpa expression in Ebf1 WT and Ebf1 KO MPP3 cells transfected with a control or a Cebpa siRNA pool. Cebpa mRNA expression relative to Actb was normalized to the control transfected samples. Data are represented as mean ± SEM. Ebf1 WT and Ebf1 KO n = 4. (E and G) Data are from >2 independent experiments.

Article Snippet: PCR products were resolved on 2% agarose gels. qRT-PCR was performed with Taqman Gene Expression Assays (FAM; Thermo Fisher Scientific) with Taqman Fast Advanced Master Mix (Thermo Fisher Scientific) using target probes against Ebf1 (Mm01288947_g1) or Cebpa (Mm01265914_s1) normalized to Act2b (Mm00607939_s1 Actb).

Techniques: RNA Sequencing, Comparison, Gene Expression, Colony-forming Unit Assay, Labeling, Expressing, Transfection, Control

Journal: The Journal of Experimental Medicine

Article Title: EBF1 primes B-lymphoid enhancers and limits the myeloid bias in murine multipotent progenitors

doi: 10.1084/jem.20212437

Figure Lengend Snippet: EBF1-dependent chromatin sites in MPP3 cells are associated with expression in CLPs. (A) Selected terms are represented from enrichment analysis of DE genes (FDR < 0.1) between Ebf1 WT and Ebf1 KO MPP3 (left) and MPP4 (right) cells. Enrichr datasets (GO_Biological_Process_2021, PanglaoDB_Augmented_2021, Gene_Perturbations_from_GEO_down) were used for enrichment analysis. Biological replicates n = 4. (B) Expression analysis of genes annotated to KO Reduced MPP3 peaks identified in ATAC analysis, in Ebf1 WT and Ebf1 KO MPP3 (upper panel), and MPP4 (lower panel) cells. The x axis represents log 10 mean normalized counts of all genes. The y axis represents shrunken |log 2 fold change| values of all genes in Ebf1 WT versus Ebf1 KO MPP3 (upper panel), and MPP4 (lower panel) cells, and is capped at 2. Genes in orange indicate the genes annotated to EBF1-dependent accessibility sites in MPP3 cells. (C) Heatmap displaying average ChIP signal for chromatin marks at DA peaks. Original data from . (D and E) Comparison of chromatin accessibility changes in MPP3 cells (D) and MPP4 (E) cells to gene expression changes in GMP versus CLP cells. The y axis represents shrunken log 2 fold changes of gene expression in GMP versus CLP cells. The x axis represents log 2 fold changes of peak read counts in Ebf1 KO versus Ebf1 WT MPP3 (D) and Ebf1 KO versus Ebf1 WT MPP4 (E) cells. Highlighted box shows positive correlation of KO Reduced MPP3 peaks and CLP gene expression. (D and E) Left: Dots represent DA peaks in MPP3 and MPP4 cells. Right: Dots represent DE genes and ATAC peaks that overlap their TSS (TSS peaks). (F and G) GSEA results of all MPP3 (F) and MPP4 (G) ATAC peaks predicted to be enhancers (CRUP probability >0.8), ranked by log 2 fold change, against a CLP-specific gene set (left) and a GMP-specific gene set (right). Positive enrichment scores reflect enrichment of the gene set in Ebf1 KO cells, negative enrichment scores reflect enrichment of the gene set in Ebf1 WT cells. CLP and GMP RNA-seq data analyzed in D–G was retrieved from GEO dataset GSE162662 .

Article Snippet: PCR products were resolved on 2% agarose gels. qRT-PCR was performed with Taqman Gene Expression Assays (FAM; Thermo Fisher Scientific) with Taqman Fast Advanced Master Mix (Thermo Fisher Scientific) using target probes against Ebf1 (Mm01288947_g1) or Cebpa (Mm01265914_s1) normalized to Act2b (Mm00607939_s1 Actb).

Techniques: Expressing, Comparison, Gene Expression, RNA Sequencing

Journal: The Journal of Experimental Medicine

Article Title: EBF1 primes B-lymphoid enhancers and limits the myeloid bias in murine multipotent progenitors

doi: 10.1084/jem.20212437

Figure Lengend Snippet: Increased myeloid bias in Ebf1 KO MPP3 and MPP4 cells is enhanced by C/EBPα. (A) Workflow of myeloid differentiation assay with cytokines (IL3, IL6, EPO, GMCSF, SCF) of MPP3 and MPP4 (upper). Frequency of CD11b + cells after 10 d of differentiation (lower). Red lines indicate the medians. Data obtained from four mice with following colony numbers: Ebf1 WT MPP3 n = 99, Ebf1 KO MPP3 n = 108, Ebf1 WT MPP4 n = 73, Ebf1 KO MPP4 n = 69. (B) Heatmap showing gene expression of DE genes associated with Cebpa KO signature identified through Enrichr enrichment analysis in . Genes are organized into MPP3-specific, common, and MPP4-specific DE genes. Genes with an annotated C/EBPα ChIP peak are labeled in blue. Heatmap scale represents z -scores calculated separately for MPP3 and MPP4 cells. (C) ChIP-seq analysis of C/EBPα occupancy in LSKs, preGM, and GMP. C/EBPα peaks are organized into MPP3-specific, common, and MPP4-specific DE genes. Regions ± 3 kb around C/EBPα summits are shown. Heatmap scale represents read coverage. Original data from ; . (D) Left: Annotated UMAP projection of a scRNA-seq landscape: mouse LK + LSK populations used as a reference for DoT score analysis; dashed area indicates the point of origin. DoT z -score values for DE genes between MPP3 Ebf1 WT versus Ebf1 KO (middle) and MPP4 Ebf1 WT versus Ebf1 KO (right). Red indicates a shift toward that cell fate and blue indicates a shift away from that cell fate. (E) Frequency of CD11b + cells after 10 d of differentiation following transfection with a control or a Cebpa siRNA pool. Red lines indicate the medians. Data obtained from four mice with following colony numbers: sictrl Ebf1 WT MPP3 n = 19, siCebpa Ebf1 WT MPP3 n = 25, sictrl Ebf1 KO MPP3 n = 36, siCebpa Ebf1 KO MPP3 n = 36. (A and E) Statistical significance was determined by Mann-Whitney U test. Data are from >2 independent experiments. Meg, megakaryocyte; Ery, erythrocyte; MC, mast cell; Bas, basophil; Neu, neutrophil; Mono/DC, monocyte/dendritic cell; Ly, lymphocyte.

Article Snippet: PCR products were resolved on 2% agarose gels. qRT-PCR was performed with Taqman Gene Expression Assays (FAM; Thermo Fisher Scientific) with Taqman Fast Advanced Master Mix (Thermo Fisher Scientific) using target probes against Ebf1 (Mm01288947_g1) or Cebpa (Mm01265914_s1) normalized to Act2b (Mm00607939_s1 Actb).

Techniques: Differentiation Assay, Gene Expression, Labeling, ChIP-sequencing, Transfection, Control, MANN-WHITNEY

Journal: The Journal of Experimental Medicine

Article Title: EBF1 primes B-lymphoid enhancers and limits the myeloid bias in murine multipotent progenitors

doi: 10.1084/jem.20212437

Figure Lengend Snippet: Ebf1 controls lymphoid/myeloid balance in HSPCs, and its loss causes increased myeloid cells in the BM. (A and C) Annotated UMAP projection of scRNA-seq landscape derived from Ebf1 WT and Ebf1 KO LK and LSK populations. Biological replicates n = 4. (B and D) Log-normalized Ebf1 expression for Ebf1 WT cells shown in A and C, respectively. (C) Cells from Nestorowa data ( ; colored) were embedded into the Ebf1 WT / Ebf1 KO landscape (gray) with annotated immunophenotypic populations. (E) Log-normalized expression of Ig and lymphoid-associated DE genes in Ebf1 WT and Ebf1 KO cells of selected clusters. Each dot represents the mean expression per mouse (four mice in total). (F) Number of DE genes between Ebf1 WT and Ebf1 KO cells per cluster. (G) DoT scores calculated using DE genes in cluster 4 between Ebf1 WT and Ebf1 KO cells in the context of the LK/LSK landscape. Mean expression in cluster 4 was used as the point of origin. Red indicates a shift toward that cell fate and blue indicates a shift away from that cell fate. (H) Violin plots showing the gene expression score of G2/M cell cycle–associated genes in Ebf1 WT and Ebf1 KO cells in selected clusters. (I) UMAP visualization of the expression of Myc and selected Myc target genes in Ebf1 WT and Ebf1 KO cells. (J) Boxplots showing the frequency of myeloid cells (CD11b + ) in the BM. Ebf1 WT n = 18, Ebf1 KO n = 16, Ighm WT and Ighm Tm1 n = 11. Statistical significance was determined by Mann–Whitney U test. Data are from >2 independent experiments. LK defined as Lin-cKit + cells. ESLAM, CD45 + EPCR + CD48 − CD150 + HSCs; Meg, megakaryocyte; Ery, erythrocyte; Neu, neutrophil; Mono/DC, monocyte/dendritic cell; Ly, lymphocyte.

Article Snippet: PCR products were resolved on 2% agarose gels. qRT-PCR was performed with Taqman Gene Expression Assays (FAM; Thermo Fisher Scientific) with Taqman Fast Advanced Master Mix (Thermo Fisher Scientific) using target probes against Ebf1 (Mm01288947_g1) or Cebpa (Mm01265914_s1) normalized to Act2b (Mm00607939_s1 Actb).

Techniques: Derivative Assay, Expressing, Gene Expression, MANN-WHITNEY

Journal: The Journal of Experimental Medicine

Article Title: EBF1 primes B-lymphoid enhancers and limits the myeloid bias in murine multipotent progenitors

doi: 10.1084/jem.20212437

Figure Lengend Snippet: Characterization of the scRNA-seq landscape in Ebf1 WT and Ebf1 KO LSK and LK cells. Related to . (A) scRNA-seq landscape derived from Ebf1 WT and Ebf1 KO LK and LSK populations, annotated for LSK and LK cells (top) and Ebf1 WT and Ebf1 KO cells (bottom). (B) UMAP visualisation of the HSC score and the expression of lineage marker genes used for the identification of cell clusters ( Procr , Dntt , Elane , Klf1 , Pf4 , Flt3, Ly6d ). (C) UMAP visualisation of the expression of Il7r and Ig genes in Ebf1 WT and Ebf1 KO cells. (D) GO enrichment analysis of the upregulated genes in cluster 4, between Ebf1 WT and Ebf1 KO cells. (E) Boxplots showing the absolute number of myeloid cells (CD11b + ) in the BM. Ebf1 WT n = 18, Ebf1 KO n = 16, Ighm WT and Ighm Tm1 n = 11. Statistical significance was determined by Mann-Whitney U test. Data are from >2 independent experiments.

Article Snippet: PCR products were resolved on 2% agarose gels. qRT-PCR was performed with Taqman Gene Expression Assays (FAM; Thermo Fisher Scientific) with Taqman Fast Advanced Master Mix (Thermo Fisher Scientific) using target probes against Ebf1 (Mm01288947_g1) or Cebpa (Mm01265914_s1) normalized to Act2b (Mm00607939_s1 Actb).

Techniques: Derivative Assay, Expressing, Marker, MANN-WHITNEY

Journal: The Journal of Experimental Medicine

Article Title: EBF1 primes B-lymphoid enhancers and limits the myeloid bias in murine multipotent progenitors

doi: 10.1084/jem.20212437

Figure Lengend Snippet: EBF1 limits C/EBPα driven myeloid bias in MPP3 and MPP4 cells. (A) Cebpa gene body and regulatory region is displayed, showing EBF1 occupancy 24 and 72 h after EBF1 induction, in pro-B cells and Hoxb8-FL cells; H3K27ac marks in pro-B cells and Hoxb8-FL cells; H3K27me3 marks in pro-B cells (original data from ; ). Chromatin accessibility in Ebf1 WT MPP3, Ebf1 KO MPP3, Ebf1 WT MPP4, and Ebf1 KO MPP4 populations. The +37 kb Cebpa enhancer is annotated, as well as the enhancer region predicted by CRUP. The scale of the y axis represents RPKM in ChIP-seq and ATAC-seq tracks. (B) Normalized read counts of Cebpa in MPP3 and MPP4 populations, in Ebf1 WT and Ebf1 KO conditions. (C) qRT-PCR analysis of Cebpa expression in Ebf1 WT MPP3 and MPP4 cells transduced with an empty or an EBF1 vector. Cebpa mRNA expression relative to Actb was normalized to the empty vector transduced samples. Data are represented as mean ± SEM. Ebf1 WT and Ebf1 KO n = 3–4. (D) Left: Annotated UMAP projection of scRNA-seq landscape derived from Ebf1 WT and Ebf1 KO LK and LSK populations. Right: UMAP visualization of Cebpa expression in Ebf1 WT and Ebf1 KO cells. (E) Representative pseudocolor plots showing the frequency of Ly6G+ neutrophils in Ebf1 WT , Ebf1 KO , Ighm WT , and Ighm Tm1 mice. (F) Boxplots showing the percentage of neutrophils in the bone marrow. Ebf1 WT and Ebf1 KO n = 19, Ighm WT and Ighm Tm1 n = 14. Statistical significance was determined by Mann-Whitney U test. (C and F) Data are from >2 independent experiments.

Article Snippet: PCR products were resolved on 2% agarose gels. qRT-PCR was performed with Taqman Gene Expression Assays (FAM; Thermo Fisher Scientific) with Taqman Fast Advanced Master Mix (Thermo Fisher Scientific) using target probes against Ebf1 (Mm01288947_g1) or Cebpa (Mm01265914_s1) normalized to Act2b (Mm00607939_s1 Actb).

Techniques: ChIP-sequencing, Quantitative RT-PCR, Expressing, Transduction, Plasmid Preparation, Derivative Assay, MANN-WHITNEY

Journal: The Journal of Experimental Medicine

Article Title: EBF1 primes B-lymphoid enhancers and limits the myeloid bias in murine multipotent progenitors

doi: 10.1084/jem.20212437

Figure Lengend Snippet: Extended analysis of the Ebf1 - Cebpa regulatory relationship. Related to . (A) Normalized read counts of Ebf1 (left) and Cebpa (right) in Ebf1 −/− pre-pro-B cells 24 and 72 h after EBF1 induction, and pro-B cells (original data from ). (B) Normalized read counts of Spi1 (PU.1) in MPP3 and MPP4 cells in Ebf1 WT and Ebf1 KO populations. (C) qRT-PCR analysis of Ebf1 expression in Ebf1 WT MPP3 and MPP4 cells transduced with an empty or an EBF1 vector. Ebf1 mRNA expression relative to Actb was normalized to the empty vector transduced samples. Data are represented as mean ± SEM. Ebf1 WT and Ebf1 KO n = 3–4. (D) Boxplots showing the absolute number of neutrophils (Ly6G + ) in the BM. Ebf1 WT n = 13, Ebf1 KO n = 14, Ighm WT and Ighm Tm1 n = 11. Statistical significance was determined by Mann-Whitney U test. (C and D) Data are from >2 independent experiments. (E) Annotated UMAP projection of scRNA-seq landscape derived from human fetal liver progenitors. (F) UMAP visualization of the expression of Procr in human fetal liver progenitors. Box indicates selected clusters (HSC/MPP, Ly/B progenitors, and Neu/Mono/DC progenitors) shown in G. (G) UMAP visualization of the expression of Ebf1 and Cebpa in selected clusters of human fetal liver progenitors. (E–G) Original data from . Meg, megakaryocyte; Ery, erythrocyte; Bas, basophil; Neu, neutrophil; Mono, monocyte; DC, dendritic cell; Ly, lymphocyte; prog, progenitors; pDC, plasmacytoid DC.

Article Snippet: PCR products were resolved on 2% agarose gels. qRT-PCR was performed with Taqman Gene Expression Assays (FAM; Thermo Fisher Scientific) with Taqman Fast Advanced Master Mix (Thermo Fisher Scientific) using target probes against Ebf1 (Mm01288947_g1) or Cebpa (Mm01265914_s1) normalized to Act2b (Mm00607939_s1 Actb).

Techniques: Quantitative RT-PCR, Expressing, Transduction, Plasmid Preparation, MANN-WHITNEY, Derivative Assay