Deciphering HoxA9 function in multipotential progenitor

biologyand B cell development

Kay L. Medina, PhDAssociate Professor

Dept. of ImmunologyMayo Clinic College of Medicine

HSC STRC

CMP

ILC2 NKP

GMP Mac PMN

MEP Ery Meg

Thymus

ETP

ELP

GMLP

ALP BLPPre-

Pro-BPro-B

Overview of adult hematopoiesis in the mouse

CDP pDC cDC

Sequentially-acting regulatory circuits orchestrate B cell fatespecification and commitment

HSC STRC

ELP

GMLP

ALP BLPPre-

Pro-BPro-B

HoxA9IkarosPU.1

Flt3E2A

E2A, Flt3

IL-7R

c-Myb

EBF1

FOXO1

IL-7R

Pax5

IL-7R

Pax5

E2A B220+CD19-

B220+CD19+

DH-JH

VH-DJH

Hoxa9 regulates Flt3 in lymphohematopoietic progenitors

Gwin and Medina, JI, 2010

hoxa9

ebf1

Inverse expression of HoxA9 and EBF1 in lymphoid/B cell precursors

CLP/BCP

Myeloid-Lymphoidprogenitor

All-Lymphoidprogenitor

B-Lymphoidprogenitor

Pro-B cell

HSC/MPP program Alternate lineage programs

B cell programTranscription factors, signaling molecules, chemokines, microRNAs

Hoxa9Flt3

Hoxa9Flt3IL7R

Hoxa9Flt3IL7

EBF1

IL7REBF1

hoxa9-/- flt3l-/- mice exhibit a severe lympho-hematopoietic block

Gwin and Medina, JI, 2013

HSC STRC GMLP

hoxa9-/- flt3l-/-

WT-RAG1-GFP+ Hoxa9-/- RAG1-GFP+ Flt3L-/- RAG1-GFP+ Hoxa9-/-Flt3L-/- RAG1-GFP+F

lt3

GFP GFP GFP GFP

ELP ELP ELP ELP

HoxA9 and Flt3 signaling function together to establish themultipotential progenitor pool competent to undergo lymphoid priming

The role of HoxA9 in B cell fate specification.

ebf1 foxo1 gfi1b

EBF1FOXO1

GFI1b

HoxA9-/- hematopoietic progenitors have impaired IL-7 signaling#

cells

x 1

05

B6 HoxA9-/- IL-7R

MF

I IL-

7R

Lin- ckit+ Flt3+

B6HoxA9

Culture model to identify HoxA9 target genes critical for B cell development

WT

HoxA9-/-

c-kit Sca-1 CD34 CD150 Flt3 IL-7R

Lin- c-kit cells cultured for 17 days in SCF+FL+Tpo+IL-6

B

# ce

lls x

106

B22

0

CD19 CD19

WT MPPs HoxA9-/- MPPs

Day 17 MPP

24 hr CLP

IL7,SCF,FL

Harvest

Switch

RNA-Seq Platform

Sequencing

WT MPP-1WT MPP-2HoxA9-/- MPP-1HoxA9-/- MPP-2

Sequencing

WT CLP-1WT CLP-2HoxA9-/- CLP-1HoxA9-/- CLP-2

2 independentexperiments

Summary of RNA-Seq Platform

Hoxa9-/- MPP

42

Hoxa9-/- MPP

73

Hoxa9-/- CLP

16

Hoxa9-/- CLP

102

13

MPP CLP

29 3 17

MPP CLP

56 86

Sample Total Reads

Hoxa9ko-CLP-1 89,662,470

Hoxa9ko-CLP-2 86,812,194

Hoxa9ko-MPP-1 79,030,174

Hoxa9ko-MPP-2 102,001,936

WT-CLP-1 108,111,316

WT-CLP-2 118,748,182

WT-MPP-1 98,958,636

WT-MPP-2 99,110,526

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-/- MPPs

64%HSC/MPP

myeloid/DC

Hoxa9-/- MPPs

54%Lymphoid

Hoxa9-/- CLPsHoxa9-/- CLPs

93%HSC/MPP

myeloid/DC

63%Lymphoid

Hoxa9-deficiency impairs lymphoid priming and downregulation of HSC/MPP and/or alternative lineage gene programs

Shared upregulated Lcn2 – lipocalin 2 – neutrophilMgam – maltase-glucoamylase - neutrophilClec4b – ATP binding cassette - DCAdam23 – disintegrin and metallopeptidase - DCPglyrp1 – peptidoglycan recognition protein - neutrophilElane – elastase – macrophage/HSC/MPPAbca13 – ATP binding cassette - neutrophilEhd3 – EH domain containing 3 – T cellsDio2 – deiodinase – HSC/MPPMpo – myeloperoxidase - monocytesVcam1 – vascular cell adhesion molecule 1 – HSC/MPPDach1 – dachshund - neutrophilPpic – peptidylprolyl isomerase C – HSC/MPPRab38 – member of RAS family - macrophageMgl2 – macrophage galactose N-acetyl-galactosamine specific Ikzf2 – ikaros zinc finger 2; Helios – HSC/T cells/T regsGata2 – HSC/MPP

Shared downregulatedRag1 – recombinase activating gene – lymphocytesGfra1 – sperm stem cell self renewal - BCPsRag2 - recombinase activating gene - lymphocytesPpfia4 – protein tyrosine phosphatase – spl DC / MacsSlc15a2 – H+/peptide transporter - BCPsP2rx3 – purinergic receptor P2X - BCPsCdh17 – cadherin 17 - BCPsCecr2 – cat eye syndrome - BCPsGimap4 – GTPase - BCPsLin28b - regulator adult to fetal HSC; let7 biogenesis - BCPsCd27 – member of TNF receptor family – MPPs/BCPsIgfbp3 – insulin growth factor 2 binding protein - BCPs

B22

0

CD19 CD19

WT MPPs HoxA9-/- MPPs

17 day MPP switched to SCF+FL+IL7 and cultured for an additional 6-8 days

Mac

-1

CD19 CD19

Gated onGFP+ cells Hoxa9-/- d17 MPP in SCF+FL+IL7

6-8 days after retroviral transduction

MigR1-GFP Mig-EBF-GFP

Ectopic expression of EBF1 is sufficient to restore the generation of BCPs from HoxA9-/- MPPs

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

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 expressionof EBF1 can bypass the IL-7 signaling defect.

Gfi1b

Runx1 IL7R

E2A* → FOXO1→ EBF1→Pax5

Flt3 → PI3KMeis1Hoxa9

MAPK

STAT5

HSC program

c-Myb Gfra1Ctr9Ccr9Scdn4

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.

Acknowledgements

Medina LabKim GwinZhihui (Cherry) Xu, MDZhixin (Jason) Hua, PhDJoe Dolence, PhDMike BellElena FrankMariya Shapiro

Flow Cytometry CoreGene Expression Core

Funding by NHLBI R01096108Eagles FoundationConcern FoundationDept of Immunology