Lymphoid to myeloid cell trans-differentiation is determined by C/EBPβ structure and post-translational modifications

Abstract

The transcription factor C/EBPβ controls differentiation, proliferation, and functionality of many cell types, including innate immune cells. A detailed molecular understanding of how C/EBPβ directs alternative cell fates remains largely elusive. A multitude of signal-dependent post-translational modifications (PTMs) differentially affect the protean C/EBPβ functions. In this study we apply an assay that converts primary mouse B lymphoid progenitors into myeloid cells in order to answer the question how C/EBPβ regulates (trans-) differentiation and determines myeloid cell fate. We found that structural alterations and various C/EBPβ PTMs determine the outcome of trans-differentiation of lymphoid into myeloid cells, including different types of monocytes/macrophages, dendritic cells, and granulocytes. The ability of C/EBPβ to recruit chromatin remodeling complexes is required for the granulocytic trans-differentiation outcome. These novel findings reveal that PTMs and structural plasticity of C/EBPβ are adaptable modular properties that integrate and rewire epigenetic functions to direct differentiation to diverse innate immune system cells, which are crucial for the organism survival.

Figure 1. Structural requirements for B cell to myeloid reprogramming potential of C/EBPβ.

Schematic representation of the different C/EBPβ constructs (left) indicating the conserved regions (CRs) in the transactivation domain (TAD; CR1,2,3,4; green, turquoise), regulatory domain (RD; CR5,6,7; red), bZip domain (yellow), and the low complexity regions (LCRs, grey). Expression of lineage specific markers: B cell CD19 (red), myeloid CD11b (blue), or double positive (magenta) at 6 (middle panel) or 9 dpi (right panel). Bar graph shows percentage of GFP⁺ gated (virus infected) cell population; B cells - control uninfected GFP^– B cell progenitors. Results represent mean ± SEM from at least two experiments.

Figure 2. C/EBPβ WT and mutants differentially regulate key myeloid genes.

RNA counts for pro-inflammatory M1, anti-inflammatory M2 and other key monocyte/macrophage genes evaluated on CD11b⁺ reprogrammed C/EBPβ ^−/− B cell progenitors. Data were calculated as log₂ and subjected to hierarchical clustering. Results represent expression profiles from three independent experiments. On the right, comparison to data obtained from reprogramming of pre-B cell line by C/EBPα is presented (Bussmann et al., 2009). MPh - WT bone marrow-derived macrophages.

Figure 3. C/EBPβ structural mutants define distinct myeloid cell trans-differentiation outcomes.

A. Representative FACS plots depicting the expression of myeloid cell markers Ly-6C, M-CSFR, and Ly-6G on 9 days trans-differentiated cells. FACS plots represent GFP⁺ CD11b⁺ cell populations, for MSCV control - GFP⁺ CD19⁺ cells. B. Distribution of the myeloid subpopulations among the reprogrammed GFP⁺ CD11b⁺ cells after staining as in A and presented as mean ± SEM. N - number of repetitions. Gr - neutrophil granulocytes, iM and rM - inflammatory and resident monocytes/macrophages, respectively, DC - dendritic cells. C. Expression of the DC markers CD11c, MHC-II and CD86 on the reprogrammed Ly-6C^– M-CSFR^– cells. Histograms represent GFP⁺ CD11b⁺ Ly-6C^– M-CSFR^– gated cells (color coded as the corresponding population on the Ly-6C/M-CSFR plot in A). “++”, “+”, “med” and “−” represent the expression levels of MHC-II and CD86 antigens. D. Cytospins of control MSCV infected CD19⁺ cells and CD11b⁺ cells reprogrammed by WT C/EBPβ or deletion mutants. B – B cells, M – macrophages, Gr – neutrophil granulocytes, * - monocytes/DCs.

Figure 4. C/EBPβ PTM site mutations affect lympho-myeloid trans-differentiation.

A. Schematic representation of C/EBPβ PTM sites and mutants tested in B-D. B. Expression of Ly-6C, M-CSFR and Ly-6G on the reprogrammed cells at 9 dpi. C. Distribution of the different myeloid populations among the reprogrammed GFP⁺ CD11b⁺ cells, stained as in B and presented as mean ± SEM. D. Cytospins of trans-differentiated sorted cells. Experiments were repeated two to three times and similar results were obtained. Gating strategies and abbreviations as in Fig. 3. E. Schematic representation of the normal hematopoiesis and lympho-myeloid reprogramming by C/EBPβ. MPP - multi potent progenitors, CLP - common lymphoid progenitor, CMP - common myeloid progenitor, GMP - granulocyte/macrophage progenitor, iMΦ and rMΦ - inflammatory and resident monocytes/macrophages.