NF-Ya activates multiple hematopoietic stem cell (HSC) regulatory genes and promotes HSC self-renewal Jiang Zhu, Yi Zhang, Gerard J. Joe, Richard Pompetti, and Stephen G. Emerson* Departments of Medicine and Pediatrics, and Abramson Cancer Center, University of Pennsylvania School of Medicine, Philadelphia, PA 19104 Communicated by Zhu Chen, Shanghai Second Medical University, Shanghai, People’s Republic of China, April 25, 2005 (received for review December 15, 2004) Hematopoietic stem cell (HSC) self-renewal and differentiation are chased from The Jackson Laboratory and maintained in the animal influenced through multiple pathways, including homeobox tran- facilities of the University of Pennsylvania with sterile water or with scription factors, signaling through ␤-catenin and Notch-1, telom- the water supplemented with neomycin sulfate and polymyxin B erase, and p27. How these multiple pathways interact and are (Sigma) within 3–4 weeks after BMT. orchestrated is currently unknown. We now report that NF-Ya, the regulatory and DNA-binding subunit of the trimeric transcription NF-Ya cDNA, MigR1 Retroviral Vector, and Preparation of Retrovi- factor NF-Y, plays a central, integrating role in several of these HSC ruses. cDNA for NF-Ya was amplified from human normal bone pathways. NF-Ya is preferentially expressed in HSC-enriched bone marrow (BM) cell RNA by using Pfu DNA polymerase (Strat- marrow subpopulations, and NF-Ya mRNA rapidly declines with agene) (7). MigR1 retroviral vector was obtained from Warren HSC differentiation. Overexpression of NF-Ya in primitive hema- Pear (Department of Pathology and Laboratory Medicine, Uni- topoietic cells activates the transcription of multiple HOX4 paral- versity of Pennsylvania). NF-Ya cDNA was directionally cloned ogs, as well as Notch-1, LEF-1, and telomerase RNA. HSCs overex- into MigR1 vector by EcoRI and XhoI sites to form plasmid pressing NF-Ya are biased toward primitive hematopoiesis in vitro MigR1-NF-Ya. For the preparation of retroviruses, the ecotropic and show strikingly increased in vivo repopulating abilities after packaging cells EcoPack2–293 (Clontech) were transiently trans- single or sequential bone marrow transplantation. Thus, NF-Ya is fected by MigR1 or MigR1-NF-Ya plasmid with Superfect (Qia- a potent cellular regulator of HSC self-renewal. gen). Between 48 and 72 h after transfection, the retroviral particle- containing supernatants were collected and filtered through SCs produce myeloid and lymphoid precursors throughout 0.45-␮m filters (Millipore) stored at Ϫ80°C before use. Hthe lifetime of the organism. To sustain hematopoiesis over months and years, hematopoietic stem cells (HSCs) have the Primitive BM Cell Isolation, Retroviral Transfection, and Primary BM unique ability to balance committed differentiation with HSC Transplantation. Primitive mouse BM cells were isolated either by Ϫ ϩ ϩϩ self-renewal. The self-renewal process, in which HSCs generate phenotype-sorting of Lin /lo Sca-1 c-Kit (LSK) BM cells or by one or two HSC daughter cells per division, is thus central for pretreating the donor mice with 5-fluorouracil (5-FU, Amersham maintaining HSC pool size throughout life. At the level of the Pharmacia and Upjohn) at a concentration of 150 mg͞kg for 4 days HSCs themselves, several genetic regulatory programs have been before BM cell harvesting. Then, 2 ϫ 106 5-FU-treated BM cells per identified to play important roles in self-renewal decisions, ml were first preincubated in 15% FBS͞DMEM (GIBCO͞BRL) including HOXB4, Notch1, Bmi-1, and the ␤-catenin pathway supplemented with 6 ng of IL-3, 10 ng of IL-6, and 100 ng of stem (1–4). Determining whether, and how, these subprograms are cell factor (SCF) per ml (R & D Systems) overnight. One milliliter coordinated at the molecular level is a central issue in under- of viral supernatant (Ϸ1 ϫ 106 viruses per ml) was added to these standing HSC biology. How these diverse intracellular biochem- cytokine-stimulated cells the next morning, along with 4 ␮g͞ml ical pathways are integrated remains largely obscure (5). Polybrene (Sigma), before the spin infection was done as described The Homeobox gene family member HOXB4 was the first by Pui (15). The infection was repeated once 24 h later. Six to 8 h transcription factor demonstrated to promote the expansion of after the second infection, the nonadherent cells were harvested HSCs both in vivo and in vitro while still retaining the HSCs’ and washed in PBS once. Then, 2 ϫ 105 live nonadherent cells were ability to differentiate into normal mature lymphoid and myeloid injected into the tail vein of one syngeneic recipient that had cells (1). Recently, we demonstrated that the trimeric transcrip- received a split 10-Gy whole-body ␥-ray irradiation. Six to 12 weeks tion factor NF-Y activates the HOXB4 promoter in cooperation after primary BMT, tissue samples were collected from the recip- with upstream͞ubiquitous stimulating factor 1 and 2 (USF1͞2) ient mice and analyzed. in normal and malignant hematopoietic cells (6, 7). Studies from other laboratories suggested that NF-Y is also a potent inducer Competitive Long-Term Lymphomyeloid Repopulation Assay. For for numerous other genes whose function likely influences stem secondary BMT, pooled BM samples from vector or NF-Ya cell (SC) function, including HOXB7, JunB, p27, CDK1, MDR1, primary recipients were sorted out into pure transduced cells by and TGF␤R-II (8–13). Such a broad role for NF-Y in early tissue GFP-activated cell sorting. Then, the vector- or NF-Ya-transduced development was also intimated by studies in Drosophila in which cells were prepared into three or four dosage levels by serial ϩ a homologue of NF-Y was shown to regulate dorsal–ventral dilutions with PBS. Next, each dosage of GFP cells was mixed with patterning (14). Thus, we reasoned that NF-Y might act as a a fixed amount of compromised (via BMT) competing B6 BM cells master gene among the network regulating HSC self-renewal and differentiation. Abbreviations: BM, bone marrow; BMT, bone marrow transplantation; ChIP, chromatin Materials and Methods immunoprecipitation; CRU, competitive repopulating units; HSC, hematopoietic stem cell; SC, stem cell; SCF, stem cell factor. a b͞ ϩ Mice. Female and male B6.SJL-Ptprc Pep3 BoyJ (SJL, CD45.1 ) ͞ ϩ *To whom correspondence should be addressed at: Division of Hematology Oncology, and C57BL͞6J (B6, CD45.2 ) mice at 8–16 weeks of age were used University of Pennsylvania School of Medicine, Maloney 510, 3600 Spruce Street, Phila- as donors͞primary recipients and secondary recipients for the bone delphia, PA 19104. E-mail: [email protected]. marrow transplantation (BMT) experiments. The mice were pur- © 2005 by The National Academy of Sciences of the USA 11728–11733 ͉ PNAS ͉ August 16, 2005 ͉ vol. 102 ͉ no. 33 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0503405102 Downloaded by guest on October 1, 2021 Fig. 1. NF-Ya overexpression induces the expression of HOX and other HSC regulatory genes in primitive hematopoietic compartment. (A) GFPϩLinϪSca-1ϩ cells were isolated by FACS from vector- or NF-Ya-trans- duced BM cells 10 weeks after primary transplanta- tion. RNA was extracted from the same amount of vector- or NF-Ya-transduced cells (1 ϫ 105), and mRNA for the indicated genes was measured by RT-PCR re- actions. (B) Four promoter regions of murine HOX4 paralogs are aligned via the conserved Y-box–E box structure. The CCAAT box-containing sequences iden- tified within the promoters of p27 and telomerase RNA gene are shown at the bottom (10, 26). The numbers in bracket indicate the distances of shown sequences from the presumed transcription start site. (C) The cobinding of NF-Y and USF1͞2 to the endoge- nous HOXB4, HOXD4, and HOXC4 promoters within leukemia cell line K562, as detected by ChIP assay. before being infused into the 10-Gy ␥-ray-irradiated B6 mice antisense 5Ј-AAATCGTCCACCTTCACCACG-3Ј; mouse (CD45.2ϩ). The secondary recipients were killed 12–16 weeks later HOXA4 primers: sense 5Ј-CCCAAGTTCCCTCCTTTCG-3Ј and for collection of hematopoietic tissues. antisense 5Ј-ATTCCTTCTCCAGTTCCAAGAC-3Ј; HOXC4 primers: sense 5Ј-GCCAGCAAGCAACCCATAGTCTAC-3Ј and CELL BIOLOGY Flow Cytometry. Freshly harvested hematopoietic tissue cells were antisense 5Ј-TCGGTGGTCCTTCTTCCATTTC-3Ј; HOXD4 suspended in 1% FCS͞PBS (GIBCO͞BRL), with or without 0.1% primers: 5Ј-GCCTACACCAGACAGCAAGTCC-3Ј and anti- NaN3 (Sigma). The nucleated cells were then stained by fluores- sense 5Ј-AAGGTCGTCAGGTCCGTATGG-3Ј; LEF-1 primers: cence-labeled antibodies (Pharmingen). The flow cytometry data sense 5Ј-GCCGACATCAAGTCATCTTTGG-3Ј and antisense were collected by using a FACScan or FACSCalibur machine 5Ј-GGGTAGAAGGTGGGGATTTCAG-3Ј; Bmi-1 primers: (Becton Dickinson) and analyzed by using FLOWJO or CELLQUEST sense 5Ј-AATGTGTGTCCTGTGTGGAGGG-3Ј and antisense software. 5Ј-TTGCTGCTGGGCATCGTAAG-3Ј; telomerase RNA prim- ers: sense 5Ј-GCTGTGGGTTCTGGTCTTTTGTTC-3Ј and 5Ј- Western Blotting. Nuclear extracts were prepared as described in ref. CGTTTGTTTTTGAGGCTCGGG-3Ј. 7. Ten micrograms of lysate was loaded onto each lane of SDS͞12% PAGE for separation and then blotted to Immobilon-P transfer In Vitro LSK Cell Culture and CFC Assay. The LSK BM cells were membranes (Millipore). The membrane was sequentially stained infected in vitro by MigR1 or MigR1-NF-Ya retroviruses by fol- with a rabbit polyclonal anti-NF-Ya IgG (Rockland, Gilbertsville, lowing the protocol of Varnum-Finney et al. (16). Then, 1 ϫ 103 PA), secondary antibodies (Santa Cruz Biotechnology), and ECL transduced cells were plated into 1 ml of 0.9% methylcellulose reagents (Amersham Pharmacia Biosciences) before the signals culture medium (StemCell Technologies, Vancouver) with 50 were developed on X-OMAT film (Kodak). ng͞ml SCF and 10 ng͞ml IL-3 and IL-6 in triplicate (17). After 7–8 days of cultivation at 37°C, the primary colonies were counted (Ͼ25 RT-PCR. Total RNAs from 1 ϫ 105 phenotypically sorted primary cells per colony). The cells retrieved from sample primary and HSCs and their progeny cells, or 1 ϫ 106 purified GFPϩ cells sorted secondary colonies were stained with Hema 3 (Fisher Scientific) for out of pooled BM samples from recipients, were isolated by using morphology examination.