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. 2016 Aug 1;197(3):910-22.
doi: 10.4049/jimmunol.1402285. Epub 2016 Jun 24.

Cooperative Activity of GABP with PU.1 or C/EBPε Regulates Lamin B Receptor Gene Expression, Implicating Their Roles in Granulocyte Nuclear Maturation

Krishnakumar Malu  1 , Rahul Garhwal  1 , Margery G H Pelletier  1 , Deepali Gotur  1 , Stephanie Halene  2 , Monika Zwerger  3 , Zhong-Fa Yang  4 , Alan G Rosmarin  4 , Peter Gaines  5
Affiliations

Cooperative Activity of GABP with PU.1 or C/EBPε Regulates Lamin B Receptor Gene Expression, Implicating Their Roles in Granulocyte Nuclear Maturation

Krishnakumar Malu et al. J Immunol. .

Abstract

Nuclear segmentation is a hallmark feature of mammalian neutrophil differentiation, but the mechanisms that control this process are poorly understood. Gene expression in maturing neutrophils requires combinatorial actions of lineage-restricted and more widely expressed transcriptional regulators. Examples include interactions of the widely expressed ETS transcription factor, GA-binding protein (GABP), with the relatively lineage-restricted E-twenty-six (ETS) factor, PU.1, and with CCAAT enhancer binding proteins, C/EBPα and C/EBPε. Whether such cooperative interactions between these transcription factors also regulate the expression of genes encoding proteins that control nuclear segmentation is unclear. We investigated the roles of ETS and C/EBP family transcription factors in regulating the gene encoding the lamin B receptor (LBR), an inner nuclear membrane protein whose expression is required for neutrophil nuclear segmentation. Although C/EBPε was previously shown to bind the Lbr promoter, surprisingly, we found that neutrophils derived from Cebpe null mice exhibited normal Lbr gene and protein expression. Instead, GABP provided transcriptional activation through the Lbr promoter in the absence of C/EBPε, and activities supported by GABP were greatly enhanced by either C/EBPε or PU.1. Both GABP and PU.1 bound Ets sites in the Lbr promoter in vitro, and in vivo within both early myeloid progenitors and differentiating neutrophils. These findings demonstrate that GABP, PU.1, and C/EBPε cooperate to control transcription of the gene encoding LBR, a nuclear envelope protein that is required for the characteristic lobulated morphology of mature neutrophils.

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Figures

FIGURE 1
FIGURE 1. Expression of Lbr during the differentiation of myeloid cell line models is not affected by the lack of C/EBPε expression
A, Northern blots that were generated from uninduced vs. differentiated myeloid cell lines and sequentially hybridized with radioactive probes generated from either Lbr or Cebpe cDNAs indicate expression levels of each gene in C/EBPε WT vs. C/EBPε KO cells. B, A Western blot identifies Lbr protein expression in either uninduced vs. ATRA induced EPRO C/EBPε WT or C/EBPε KO cells. C, A Northern blot generated from total RNA extracted from uninduced or induced SCF ER-Hoxb8 cells and sequentially hybridized with Lbr, Cebpe, and Spi1 cDNA probes identifies changes in gene expression patterns. The bottom panels of each figure indicate either transcription levels of the gene encoding β-actin (Actb) or protein expression to demonstrate amounts of either RNA or protein loaded into each lane, respectively. Data for expression of Cebpe and Actb in A and C are reprinted from Halene et al., 2010 (see reference 70).
FIGURE 2
FIGURE 2. Expression of PU.1 and C/EBPε increase during myeloid differentiation whereas GABPα expression is highly expressed at multiple stages
A, Western blots generated from protein lysates of mouse myeloid cells as either EML progenitors or derived EPRO cells, COS-1 fibroblasts, and HEK293 fibroblasts (left panels), or undifferentiated vs. differentiated human myeloblastic HL-60 cells (right panels), were sequentially probed using Abs against GABPα or β-actin as a loading control. B, The original EML cells and SCF ER-Hoxb8 were induced to mature neutrophils in order to extract total protein for two additional Western blot assays. Shown in the top panels are images of Wright-Giemsa-stained cells from three different stages of EML/EPRO cell differentiation and either uninduced (D0) or differentiated (D5) SCF ER-Hoxb8 cells, in order to demonstrate nuclear morphologic maturation of cells from each induction. Western blots were then generated from these cells (as well as ATRA-induced D1 EPRO cells) that were sequentially probed with anti-GABPα, PU.1, or C/EBPε Abs, then with anti-tubulin Ab to demonstrate amounts of protein loaded in each lane. C, Real-time PCR assays were performed on reverse transcribed cDNAs of the transcription factors GABP, PU.1, C/EBPε, and C/EBPα, each amplified using total RNA extracted from bone marrow-derived primary HSC cultured ex vivo in SCF plus IL-3 for three days (D3), then SCF/IL-3 plus G-CSF for two days (D5), and finally G-CSF alone for two days (D7). Shown in the top panels are images of Wright-Giemsa-stained cells that were used to extract total RNA. The results of the real-time PCR amplifications are depicted in two graphs: the upper panel indicates fold-changes in amplification of each gene when compared to levels in the myeloid progenitors (IL-3/SCF D3), whereas those shown in the bottom panel compare transcription levels of each factor to those of GABPα at each stage of differentiation. All fold-differences were normalized to the levels of mS18 transcription as a reference standard, and are averages ± SD from three independent assays.
FIGURE 3
FIGURE 3. Activation of the Lbr promoter by GABP plus PU.1 is mediated by the proximal and distal Ets sites
A, Depicted is a schematic representation of the mouse Lbr promoter that includes a diagram of the two identified Ets sites (open arrowheads) with respect to the putative TSS and the putative C/EBP binding sites (indicated by shaded and open boxes). Shown beneath this diagram are the sequences of the proximal and distal Ets sites with the conserved GAGGAA core sequences and their locations in the promoter, and a depiction of the two promoter constructs used in the in vitro reporter assays. B, Luciferase activities supported by either the proximal Ets sites (pGL3-Lbr (-744)) or both these and the distal Ets site (pGL3-Lbr (-1513)) were assessed in transfected COS-1 cells. Shown are fold activities of relative luciferase levels driven by the two different sized promoter sequences or those with mutated Ets binding sites (GGAA sequences were changed to CCAA, indicated by the X through arrowheads that depict each Ets site), each inserted into the pGL3-Basic vector and compared to levels produced by the empty vector. Ectopic expression of the indicated transcription factors in the transfected COS-1 cells were provided by either the pCMV vector (GABPα, GABPβ and C/EBPε) or the pMSCV vector (C/EBPα and PU.1). C and D, Increases in luciferase activities upon expression of the different combinations of transcription factors were identified in COS-1 cells co-transfected with the pGL3-Lbr (-1513) reporter vector along with expression vectors for each of the indicated transcription factors (the combination of expression vectors for both GABPα and GABPβ in the same transfections is indicated by GABPα/β). All levels of normalized luciferase activities were used to calculate the indicated fold-activation levels as compared to cells transfected with the reporter vector plus an empty pCMV vector. E, Use of the pGL3-Lbr (-1513) reporter vector with mutated Lbr promoter sequences identify decreased activities in response to ectopically expressed GABP plus PU.1 as compared to WT sequences. All values shown in B-E are averages of fold increases in relative luciferase activities (each normalized to control β-galactosidase levels) ± SD from three independent assays.
FIGURE 4
FIGURE 4. GABPα binds to Ets sites in the Lbr promoter
A, Shown are the sequences used to generate probes in the EMSAs, depicting the location of each probe with respect to the consensus GGAA sites (underlined and bold) in either binding location. B, EMSAs performed using nuclear lysates from HEK293 cells transfected with expression vectors for both GABPα/β and PU.1 identify shifted bands created by 32P-radiolabeled proximal Ets probes (lanes 2 – 7, left block arrows) but not by the labeled probe alone (lane 1). Addition of 100X excess cold competitor disrupted the formation of the lower bands whereas addition of an irrelevant probe had no affect (lanes 3 and 4, respectively). Inclusion of anti-GABPα Abs caused one of the shifted bands (black filled arrow) to become supershifted (lanes 5–7, with supershifted species indicated by the top arrow), but did not affect a second, higher mobility species (shaded block arrow), nor did it affect a less intense, lower mobility species (open block arrow).
FIGURE 5
FIGURE 5. Binding of GABP to the proximal Ets sites is disrupted by either anti-GABPα or PU.1 antibodies, or mutation of the consensus Ets sequence
EMSAs performed using nuclear lysates from HEK293 cells transfected with GABPα, GABPβ, and PU.1 (upper panel) with the proximal Ets probe identify three high molecular weight species, two of which (open and filled block arrows) were supershifted by including either anti-GABPα or anti-PU.1 Abs (lanes 6 and 7, respectively). The addition of anti-PU.1 Ab also caused less formation, and therefore diminished the intensity of the smallest band (indicated by shaded block arrow, lane 7), whereas neither IgG (lane 5) nor anti-C/EBPε Ab (lane 8) affected the shifted bands. Use of probes with GGAA to CCAA mutations at the Ets sites (distal indicated by Dist-Mut, proximal by Prox-Mut, and both sites by Prox+Dist-Mut) disrupted formation of the two most prominent bands, but seemed to increase intensities of the higher molecular weight species (open block arrow). The mutant forms did not change amounts of probe that remained in the wells (arrow). The lower panel indicates EMSAs that utilized the same probes with EML nuclear lysates, in which 3 less intense bands were identified. Two higher mobility species (indicated by the closed and shaded block arrows) were abolished upon use of cold competitor (lower panel, lane 3), and were diminished by inclusion of anti-GABPα or anti-PU.1 Abs (lower panel, lanes 6 and 7, respectively). Use of the mutant Ets sites in the probes also disrupted formation of the two most prominent bands, but did not appear to affect the formation of the higher molecular weight species except for use of Dist-Mut (bands indicated by the open block arrow).
FIGURE 6
FIGURE 6. In vivo binding of GABPα and PU.1 to both proximal and distal Ets sites
A, Shown are the sequences spanning the proximal and distal Ets sites plus the Cebp site and primers used for the PCR assays in the ChIP experiments (underlined), each with regards to the consensus binding sites (shown in bold). B, ChIP assays performed using nuclear lysates from EML cells, Abs against GABPα or PU.1, and the proximal (B) or distal (C) primer sets, which produced fragments that were visualized with a 0.9% agarose gel (upper panel, also shown are the molecular weight marker (MWM) bands and products of reactions with either no Ab, input DNA, or no input DNA (Neg)). The assays were also performed using real-time PCR to identify the percentage of amplified DNA precipitated by the indicated Abs to that amplified from input DNA, using the same reaction mixtures with Abs for GABP and PU.1 but also reactions that identified binding of C/EBPε to the identified Cebpe site (lower panel). C, The same reactions were performed using nuclear extracts from uninduced EPRO cells and ATRA-induced EPRO cells after either 3 or 5 days of induction. Shown are percentages of amplified DNA as compared to input DNA for each of the binding sites and their corresponding transcription factors. Data shown for each real-time PCR result are averages ± SD from three independent assays. All differences between amplified products precipitated by GABPα, PU.1 or C/EBPε vs. IgG were statistically significant with p values less than 0.0001, with the exception of that indicated in C by an asterisk (p = 0.0043).
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