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Deciphering HoxA9 function in multipotential progenitor biology and B cell development

This study explores the role of HoxA9 in multipotential progenitor biology and B cell development. It investigates the sequential regulatory circuits involved in B cell fate specification and commitment, as well as the function of HoxA9 and Flt3 in establishing the multipotential progenitor pool. The study shows that HoxA9 deficiency impairs lymphoid priming and downregulation of HSC/MPP and alternative lineage gene programs. Enforced expression of EBF1 can bypass the IL-7 signaling defect caused by HoxA9 deficiency.

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Deciphering HoxA9 function in multipotential progenitor biology and B cell development

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  1. Deciphering HoxA9 function in multipotential progenitor biology and B cell development Kay L. Medina, PhD Associate Professor Dept. of Immunology Mayo Clinic College of Medicine

  2. Overview of adult hematopoiesis in the mouse PMN Mac GMP Meg Ery MEP CMP cDC pDC CDP HSC STRC GMLP Pre- Pro-B Pro-B BLP ELP ALP ILC2 NKP Thymus ETP

  3. Sequentially-acting regulatory circuits orchestrate B cell fate specification and commitment B220+ CD19+ B220+ CD19- HoxA9 Ikaros PU.1 Flt3 E2A E2A HSC STRC GMLP E2A, Flt3 DH-JH Pre- Pro-B IL-7R Pax5 IL-7R Pax5 IL-7R c-Myb EBF1 FOXO1 Pro-B VH-DJH BLP ELP ALP

  4. Hoxa9 regulates Flt3 in lymphohematopoietic progenitors Gwin and Medina, JI, 2010

  5. Inverse expression of HoxA9 and EBF1 in lymphoid/B cell precursors hoxa9 ebf1 CLP/BCP

  6. HSC/MPP program Alternate lineage programs Hoxa9 Flt3 IL7 EBF1 Hoxa9 Flt3 IL7R IL7R EBF1 Hoxa9 Flt3 Pro-B cell B-Lymphoid progenitor All-Lymphoid progenitor Myeloid- Lymphoid progenitor Transcription factors, signaling molecules, chemokines, microRNAs B cell program

  7. hoxa9-/- flt3l-/- mice exhibit a severe lympho-hematopoietic block Gwin and Medina, JI, 2013

  8. HoxA9 and Flt3 signaling function together to establish the multipotential progenitor pool competent to undergo lymphoid priming Hoxa9-/-Flt3L-/- RAG1-GFP+ WT-RAG1-GFP+ Hoxa9-/- RAG1-GFP+ Flt3L-/- RAG1-GFP+ GMLP ELP ELP ELP ELP Flt3 STRC HSC hoxa9-/- flt3l-/- GFP GFP GFP GFP

  9. The role of HoxA9 in B cell fate specification. EBF1 FOXO1 GFI1b ebf1 foxo1 gfi1b

  10. HoxA9-/- hematopoietic progenitors have impaired IL-7 signaling Lin- ckit+ Flt3+ B6 HoxA9 MFI IL-7R # cells x 105 B6 HoxA9-/- IL-7Ra

  11. Culture model to identify HoxA9 target genes critical for B cell development Lin- c-kit cells cultured for 17 days in SCF+FL+Tpo+IL-6 WT HoxA9-/- CD34 Flt3 c-kit Sca-1 CD150 IL-7R B WT MPPs HoxA9-/- MPPs B220 # cells x 106 CD19 CD19

  12. RNA-Seq Platform Day 17 MPP 24 hr CLP IL7,SCF,FL Harvest Switch 2 independent experiments Sequencing WT MPP-1 WT MPP-2 HoxA9-/- MPP-1 HoxA9-/- MPP-2 Sequencing WT CLP-1 WT CLP-2 HoxA9-/- CLP-1 HoxA9-/- CLP-2

  13. Summary of RNA-Seq Platform Read length:50bp 40-50X depth of coverage/gene Differential expression cutoff: absolute fold change 1.4 False discovery rate of ≥0.1; p>0.05 Hoxa9-/- CLP 16 Hoxa9-/- MPP 42 Hoxa9-/- MPP 73 Hoxa9-/- CLP 102 MPP CLP MPP CLP 3 29 13 56 17 86

  14. Hoxa9-deficiency impairs lymphoid priming and downregulation of HSC/MPP and/or alternative lineage gene programs Hoxa9-/- CLPs Hoxa9-/- CLPs Hoxa9-/- MPPs Hoxa9-/- MPPs 64% HSC/MPP myeloid/DC 63% Lymphoid 54% Lymphoid 93% HSC/MPP myeloid/DC Shared upregulated Lcn2 – lipocalin 2 – neutrophil Mgam – maltase-glucoamylase - neutrophil Clec4b – ATP binding cassette - DC Adam23 – disintegrin and metallopeptidase - DC Pglyrp1 – peptidoglycan recognition protein - neutrophil Elane – elastase – macrophage/HSC/MPP Abca13 – ATP binding cassette - neutrophil Ehd3 – EH domain containing 3 – T cells Dio2 – deiodinase – HSC/MPP Mpo – myeloperoxidase - monocytes Vcam1 – vascular cell adhesion molecule 1 – HSC/MPP Dach1 – dachshund - neutrophil Ppic – peptidylprolyl isomerase C – HSC/MPP Rab38 – member of RAS family - macrophage Mgl2 – macrophage galactose N-acetyl-galactosamine specific Ikzf2 – ikaros zinc finger 2; Helios – HSC/T cells/T regs Gata2 – HSC/MPP Shared downregulated Rag1 – recombinase activating gene – lymphocytes Gfra1 – sperm stem cell self renewal - BCPs Rag2 - recombinase activating gene - lymphocytes Ppfia4 – protein tyrosine phosphatase – spl DC / Macs Slc15a2 – H+/peptide transporter - BCPs P2rx3 – purinergic receptor P2X - BCPs Cdh17 – cadherin 17 - BCPs Cecr2 – cat eye syndrome - BCPs Gimap4 – GTPase - BCPs Lin28b - regulator adult to fetal HSC; let7 biogenesis - BCPs Cd27 – member of TNF receptor family – MPPs/BCPs Igfbp3 – insulin growth factor 2 binding protein - BCPs

  15. Ectopic expression of EBF1 is sufficient to restore the generation of BCPs from HoxA9-/- MPPs HoxA9-/- MPPs WT MPPs 17 day MPP switched to SCF+FL+IL7 and cultured for an additional 6-8 days B220 CD19 CD19 MigR1-GFP Mig-EBF-GFP Gated on GFP+ cells Hoxa9-/- d17 MPP in SCF+FL+IL7 6-8 days after retroviral transduction Mac-1 CD19 CD19

  16. Linking RNA-Seq and Microarray data to the IL-7R signaling pathway

  17. Conclusions: We developed a short-term in vitro culture model to expand MPPs from WT and HoxA9-/- mice that will be informative in the identification, characterization, and validation of HoxA9 regulated genetic circuits in early hematopoiesis. RNA-Seq confirmed decreased lymphoid priming in MPPs and under B cell differentiation conditions due to HoxA9 deficiency independent of Flt3 signaling. HoxA9 deficiency impairs IL-7R expression and signaling – enforced expression of EBF1 can bypass the IL-7 signaling defect. Gfi1b Runx1 IL7R E2A* → FOXO1→ EBF1→Pax5 Flt3 → PI3K STAT5 c-Myb HSC program Meis1 Hoxa9 MAPK Gfra1 Ctr9 Ccr9 Scdn4

  18. Underway: 1. Identify novel HoxA9 target genes : compare results to microarray platform of genes upregulated in EBF1-/- multipotent cells that express endogenous Hoxa9 - underexpressed in Hoxa9-/- MPPs in RNA-Seq platform (transcriptional activation) - overexpressed in EBF1-/- MPPs in Affymetrix Array Chd17, CD27, CCR9, Ppfia4, Sorcs2, RAG2, GPR25, Jup, Igf2bp3 - overexpressed in Hoxa9-/- MPPs in RNA-Seq platform (transcriptional repressor) - underexpressed in EBF1-/- MPPs in Affymetrix Array Mgst1, Tgm2, Enpp4, Plekha8, Muc13, Cpne2, Tmem176a, Mreg, Adcy6, STAT4, Sulf2, SpiB 2. Identify novel EBF1 target genes : compare results to microarray platform of genes underexpressed in EBF1-/- multipotent cells to genes underexpressed in HoxA9-deficient MPPs or CLPs P2rx3, RAG1, Rgs8, Cecr2, Egfl7, Uaca, Lax1, Dntt, Slc15a2, Gimap4 3. Perform gain-of-function and loss-of-function experiments in WT or HoxA9-/- MPPs to evaluate gene function in B cell development followed by molecular validation. 4. Determine if HoxA9 target genes are relevant to leukemogenesis.

  19. Acknowledgements Flow Cytometry Core Gene Expression Core Funding by NHLBI R01096108 Eagles Foundation Concern Foundation Dept of Immunology Medina Lab Kim Gwin Zhihui (Cherry) Xu, MD Zhixin (Jason) Hua, PhD Joe Dolence, PhD Mike Bell Elena Frank Mariya Shapiro

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