Leukemia (2014) 28, 1676–1686 & 2014 Macmillan Publishers Limited All rights reserved 0887-6924/14 www.nature.com/leu ORIGINAL ARTICLE Dual regulation of SPI1/PU.1 transcription factor by heat shock factor 1 (HSF1) during macrophage differentiation of monocytes G Jego1,2, D Lanneau1,2, A De Thonel1,2, K Berthenet1,2, A Hazoume´ 1,2, N Droin3,4, A Hamman1,2, F Girodon1,2, P-S Bellaye1,2, G Wettstein1,2, A Jacquel1,2,5, L Duplomb6,7, A Le Moue¨l8,9, C Papanayotou10, E Christians11, P Bonniaud1,2, V Lallemand-Mezger8,9, E Solary3,4 and C Garrido1,2,12 In addition to their cytoprotective role in stressful conditions, heat shock proteins (HSPs) are involved in specific differentiation pathways, for example, we have identified a role for HSP90 in macrophage differentiation of human peripheral blood monocytes that are exposed to macrophage colony-stimulating factor (M-CSF). Here, we show that deletion of the main transcription factor involved in heat shock gene regulation, heat shock factor 1 (HSF1), affects M-CSF-driven differentiation of mouse bone marrow cells. HSF1 transiently accumulates in the nucleus of human monocytes undergoing macrophage differentiation, including M-CSF- treated peripheral blood monocytes and phorbol ester-treated THP1 cells. We demonstrate that HSF1 has a dual effect on SPI1/ PU.1, a transcription factor essential for macrophage differentiation and whose deregulation can lead to the development of leukemias and lymphomas. Firstly, HSF1 regulates SPI1/PU.1 gene expression through its binding to a heat shock element within the intron 2 of this gene. Furthermore, downregulation or inhibition of HSF1 impaired both SPI1/PU.1-targeted gene transcription and macrophage differentiation. Secondly, HSF1 induces the expression of HSP70 that interacts with SPI1/PU.1 to protect the transcription factor from proteasomal degradation. Taken together, HSF1 appears as a fine-tuning regulator of SPI1/PU.1 expression at the transcriptional and post-translational levels during macrophage differentiation of monocytes. Leukemia (2014) 28, 1676–1686; doi:10.1038/leu.2014.63 INTRODUCTION The transcription factor SPI1/PU.1 is a member of the Ets family 8,9 Exposure to a wide variety of physical and chemical stresses proteins expressed in myeloid and B lymphoid cells. It has an activates the expression of stress response genes coding for heat essential role in the acquisition of the macrophage phenotype by shock proteins (HSPs). HSPs are molecular chaperones that help regulating the expression of many myeloid genes, such as those the cell to cope with these stressful conditions by mediating encoding the receptor of the macrophage colony-stimulating correct refolding of the denatured proteins. Besides their well- factor (M-CSFR, also known as CSF1R), the hemoglobin scavenger described role in cell protection under stressful conditions, HSPs receptor CD163, the alpha-M integrin molecule CD11b and the have essential roles in a variety of cellular processes, such as cell cytokine interleukin-1b (IL-lb). The level of SPI1/PU.1 is critical in cycle and apoptosis.1 HSPs have also demonstrated essential specifying cell fate, as its deregulation can lead to the 9–11 functions in cell differentiation, for example, in erythroblast2,3 and development of leukemias and lymphomas. Previous reports macrophage differentiation,4 and their level of expression is demonstrated that the proximal promoter of human SPI1/PU.1 tightly regulated during development.5 gene and distal regulatory elements located at À15, À14 and Expression of heat shock genes is regulated by heat shock À12 kb upstream of the transcription start site, were essential for 12,13 transcription factors (HSFs), which bind to heat shock elements (HSE) SPI1/PU.1 gene regulation. In the present study, we identify a in their promoter region and stimulate their transcription.5,6 dual function of HSF1 in monocyte to macrophage differentiation: Four members have been identified in mammals: HSF1, HSF1 regulates SPI1/PU.1 gene expression, and also concomitantly HSF2, HSF3 and HSF4. HSF1 is the major stress-responsive the expression of Hsp70 gene; the resulting newly synthesized family member. In response to stressful stimuli, HSF1 is HSP70 protein interacts with SPI1/PU.1 protein to prevent its activated by trimerization and hyperphosphorylation.5 As a proteasomal degradation. result, HSF1 binds to HSE and activates the transcription of heat shock genes, which results in the accumulation of HSPs such as HSP70 with, as a final outcome, cell protection. HSF1 not only MATERIALS AND METHODS regulates expression of heat shock genes in response to stress Mice and monocyte purification but it is also involved in development, by regulating non-heat The Hsf1-knockout mouse line was derived from animals created by shock genes.7 homologous recombination with a gene-targeting vector in embryonic 1INSERM, UMR 866, ‘Equipe Labellise´e Ligue contre le Cancer’, Dijon, France; 2Faculty of Medicine and Pharmacy, University of Burgundy, Dijon, France; 3INSERM, UMR 1009, Institut Gustave Roussy, 114 rue Edouard Vaillaint, Villejuif, France; 4University Paris-Sud 11, Institut Gustave Roussy, 114 rue Edouard Vaillaint, Villejuif, France; 5INSERM, U526, Nice, France; 6Faculty of Medicine and Pharmacy, Ge´ne´tique et anomalies du de´veloppement, University of Burgundy, Dijon, France; 7CHU, Dijon, France; 8CNRS, UMR7216 E´pige´ne´tique et Destin Cellulaire, 35 rue He´le`ne Brion, Paris, France; 9University Paris Diderot, Sorbonne Paris Cite´, 35 rue He´le`ne Brion, Paris, France; 10University Paris Diderot, Sorbonne Paris Cite´, Institut jacques Monod, UMR 7592, Paris cedex 13, France; 11CNRS, UMR 5547, Universite´ Paul Sabatier, 118 route de Narbonne, Toulouse, France and 12Centre de lutte contre le cancer George-Franc¸ois Leclerc, Dijon, France. Correspondence: Professor C Garrido, Faculty of Medicine and Pharmacy, INSERM UMR 866, ‘Equipe Labellise´e Ligue contre le Cancer’, 7 boulevard Jeanne D’Arc, Dijon 21079, France. E-mail: [email protected] Received 2 September 2013; revised 24 January 2014; accepted 27 January 2014; accepted article preview online 7 February 2014; advance online publication, 6 May 2014 HSF1 controls SPI1/PU.1 expression G Jego et al 1677 stem cells, described by McMillan et al.,14 Hsf1-knockout mice were bred Immunofluorescence staining and maintained in a mixed genetic background C57BL/6 J Â BALB/c, Staining was done as previously described.4 After fixation and À / À þ / À allowing the production of 15% viable Hsf1 by crossing Hsf1 mice. permeabilization, cells were incubated overnight at 4 1C with anti-HSP70 So far, only one study on the lymphoid lineage in spleen, thymus and bone (Tebu-Bio, Le Perray-en-Yvelines, France), anti-SPI1/PU.1 (Ozyme), anti- À / À marrow from hsf1 mice has been performed with no phenotypic HSF1 (Tebu-Bio) before incubation with secondary antibodies (Molecular 15 outcome. Experiments were performed with the approval of the Ethics Probe, Leiden, the Netherlands). The nucleus was labeled by 4’,6- committee of the University of Burgundy. Bone marrow cells were diamidino-2-phenylindole (Sigma-Aldrich). Images were acquired using À / À extracted from tibias and femurs of wild-type and Hsf1 mice, labeled the Cell Observer station (Zeiss, Germany). with fluorescein isothiocyanate -labeled anti-CD49b, -CD45R, -CD3e and -ter119 antibodies (Miltenyi Biotec, Paris, France), washed and incubated Chromatin immunoprecipitation (ChIP) with anti-fluorescein isothiocyanate microbeads (Miltenyi Biotec). Myeloid ChIP experiments were performed using Millipore EZ-ChIP ChIPKit cells were then purified with the Automacs and treated with murine 6 M-CSF (10 ng/ml, R&D Systems, Abington, UK). The percentage of CD11b þ / according to the manufacturer’s instructions. Cells (50 Â 10 ) were first CD3 À /B220 À myeloid cells, CD19 þ cells, Ter119 cells and Lin À Sca1 þ fixed by 37% formaldehyde. Glycine was added and cells were lysed in SDS c-kit þ in the bone marrow, and CD3 þ cells and B220 þ cells in the blood lysis buffer after two washes with PBS. Samples were sonicated (10 pulses were determined by flow cytometry. The percentage of macrophages after of 30 s, Bioruptor’s Diagenode, Diagenode, Belgium) and immunoprecipi- tation of crosslinked protein/DNA was performed with an anti-HSF1 4 days of culture was determined by Gr1 À F4/80 þ staining. Untouched (Ozyme), an anti-dimethyl histone (positive control, Ozyme) or a normal human monocytes were purified from buffy coats using the monocyte isolation kit II (Miltenyi Biotec). mouse IgG antibody. The complexes were washed five times. After reverse crosslinking, the DNA was purified. Primers for the PCR are available in Supplementary methods. For the experiment shown in Figure 1f, ChIP experiments were performed using ChIP-IT (ActiveMotif, La Hulpe, Cell culture and differentiation Belgium). Chromatin, immunoprecipitated by HSF1 (ThermoFisher Human monocytes were isolated from healthy and chronic myelomono- Scientific, Fremont, CA, USA), phospho Ser5pol II (Active Motif), histone cytic leukemia (CMML) patients blood samples (IIGR, Villejuif, CHU, Dijon, H3K27Ac (Abcam) or SPI1/PU.1 antibodies (Ozyme) was eluted from France), after obtaining a written consent. HeLa, HEK293 T and THP1 cells the magnetic beads after several washes and the crosslinks of these (DSMZ, Braunschweig, Germany), were cultured in RPMI 1640 medium sequentially immunoprecipitated protein–DNA complexes were then supplemented with 10% fetal bovine serum. THP1 cells were induced