Microarray profiling of Gfi1b and Meis1 expression in erythroid (MEL) cells upon LSD1 inhibition.

<p>Increase in mRNA levels of Gfi1b and Meis1 (fold increase) in LSD1 depleted MEL cells relative to controls.</p

Differential Transcriptional Regulation of <em>meis1</em> by Gfi1b and Its Co-Factors LSD1 and CoREST

<div><p>Gfi1b (growth factor independence 1b) is a zinc finger transcription factor essential for development of the erythroid and megakaryocytic lineages. To elucidate the mechanism underlying Gfi1b function, potential downstream transcriptional targets were identified by chromatin immunoprecipitation and expression profiling approaches. The combination of these approaches revealed the oncogene <em>meis1</em>, which encodes a homeobox protein, as a direct and prominent target of Gfi1b. Examination of the <em>meis1</em> promoter sequence revealed multiple Gfi1/1b consensus binding motifs. Distinct regions of the promoter were occupied by Gfi1b and its cofactors LSD1 and CoREST/Rcor1, in erythroid cells but not in the closely related megakaryocyte lineage. Accordingly, Meis1 was significantly upregulated in LSD1 inhibited erythroid cells, but not in megakaryocytes. This lineage specific upregulation in Meis1 expression was accompanied by a parallel increase in di-methyl histone3 lysine4 levels in the Meis1 promoter in LSD1 inhibited, erythroid cells. Meis1 was also substantially upregulated in <em>gfi1b−/−</em> fetal liver cells along with its transcriptional partners Pbx1 and several Hox messages. Elevated Meis1 message levels persisted in <em>gfi1b</em> mutant fetal liver cells differentiated along the erythroid lineage, relative to wild type. However, cells differentiated along the megakaryocytic lineage, exhibited no difference in Meis1 levels between controls and mutants. Transfection experiments further demonstrated specific repression of <em>meis1</em> promoter driven reporters by wild type Gfi1b but neither by a SNAG domain mutant nor by a DNA binding deficient one, thus confirming direct functional regulation of this promoter by the Gfi1b transcriptional complex. Overall, our results demonstrate direct yet differential regulation of <em>meis1</em> transcription by Gfi1b in distinct hematopoietic lineages thus revealing it to be a common, albeit lineage specific, target of both Gfi1b and its paralog Gfi1.</p> </div

Message levels of Meis1, Pbx1 and Hox family members in fetal liver cells.

<p><b>A.</b> QPCR of relative Gfi1b and Meis1 mRNA levels (normalized for HPRT) in embryonic day 12.5 (e12.5) wild type, <i>gfi1b+/−</i> and <i>gfi1b−/−</i> fetal liver cells. <b>B.</b> QPCR of relative Meis1 mRNA levels (normalized for HPRT) in control (wild type or <i>gfi1b+/−</i>) and <i>gfi1b−/−</i> fetal liver cells differentiated along the erythroid or megakaryocytic lineages <i>ex-vivo</i>. <b>C.</b> QPCR of relative Hoxa9, Pbx1, Hoxb3, Hoxc4 and Hoxd13 message levels from total fetal liver cells of the indicated genotypes. Averages and standard deviations from multiple embryos of different genotypes from three cohorts are shown. The p values for Pbx1, Hoxb3 and Hoxc4 levels in wild type versus mutant cells were <0.001 and those for Hoxa9 and Hoxd13 were <0.01. All values <0.05 were considered significant.</p

Regulation of the isolated <i>meis1</i> promoter by exogenous Gfi1b.

<p>Luciferase reporter based promoter assays in HEK-293T cells. 1 µg of reporter plasmid along with the indicated amount of expression plasmid and 50 ng of β-galactosidase expression vector was transfected into ∼10⁶ cells and assayed for luciferase activity. The 0.5 kb <i>gfi1b</i> core promoter was used as a positive control. The <i>meis1L</i> promoter consisted of 2.7 kb of promoter sequence from −2.1 kb to +0.65 kb (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053666#pone-0053666-g002" target="_blank">Figure 2A</a>) and the <i>meis1S</i> consisted of 1.25 kb of promoter sequence from −0.60 kb to +0.65 kb relative to the tss of <i>meis1</i>. P2A-represents the P2A-Gfi1b SNAG domain mutant; Gfi1b-del5+6-represents the Gfi1b deletion mutant lacking zinc fingers 5 and 6. For each promoter set, values shown are relative to that obtained in the absence of the corresponding expression vector, following normalization of all luciferase values for β-galactosidase levels. The average (solid bars) and standard deviations (error bars) from 3 independent experiments is shown.</p

Message levels of Gfi1b and Meis1 in erythroid and megakaryocytic cells.

<p>Quantitative PCR (qPCR) of relative Gfi1b and Meis1 mRNA levels (normalized for HPRT) in (A) MEL (erythroid) and (B) L8057 (megakaryocytic) cells transduced with empty vector (mIR-PIG) or LSD1 shRNA (LSD1 k/d). Average of three experiments is shown, error bars represent standard deviation. C. Steady state protein levels of Gfi1b, LSD1 and CoREST relative to β-actin in HEK-293T, MEL and L8057 cells.</p

Occupancy of two distinct promoter regions of the <i>meis1</i> promoter by Gfi1b/LSD1/CoREST.

<p><b>A.</b> 2.1 kb sequence of the murine Meis1 promoter and ∼0.75 kb of coding sequence spanning the two ∼1.2 kb segments (denoted in black uppercase lettering) obtained from ChIP on chip screening for Gfi1b/LSD1/CoREST targets. Intervening and downstream sequences not obtained from ChIP are indicated in grey lowercase letters. The putative transcriptional start site (indicated as +1 in the sequence) as inferred from the <i>meis1</i> transcript sequence (NM_010789) reported in the nucleotide database (also see text) and the initiator codon, (also according to the database) are indicated in green. The Gfi1b consensus elements are highlighted in bold maroon lettering. Sequences of primers used for ChIP qPCR or for amplification of promoter segments for subcloning are underlined (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053666#s2" target="_blank">Materials and Methods</a>). <b>B,C.</b> Chromatin immunoprecipitation (ChIP) analysis of the Gfi1b promoter and two Meis1 promoter segments (Meis1.1: distal; relative to tss and Meis1.2: promoter proximal) in erythroid and megakaryocytic lineages. Enrichment of the indicated promoter sequences relative to the immunoglobulin switch µ (Sµ) sequence in MEL (B) and L8057 (C) cells are indicated. Results shown are the average (solid bars) and standard deviations (error bars) of three independent experiments.</p

Di-methyl H3–K4 levels in <i>meis1</i> promoter chromatin.

<p>Chromatin immunoprecipitation (ChIP) analysis of the <i>gfi1b</i> promoter and two <i>meis1</i> promoter segments as indicated <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053666#pone-0053666-g002" target="_blank">Figure 2A</a> in erythroid and megakaryocytic lineages. Relative enrichment of the indicated promoter sequences for di-methyl H3–K4 in control (scrambled) versus LSD1 knocked down cells was calculated relative to that for immunoglobulin switch µ (Sµ) sequences in MEL (A) and L8057 (B) cells respectively. Results shown are the average (solid bars) and standard deviations (error bars) of three independent experiments.</p

Cooperative Stimulation of Megakaryocytic Differentiation by Gfi1b Gene Targets Kindlin3 and Talin1

<div><p>Understanding the production and differentiation of megakaryocytes from progenitors is crucial for realizing the biology and functions of these vital cells. Previous gene ablation studies demonstrated the essential role of the transcriptional repressor Gfi1b (growth factor independence 1b) in the generation of both erythroid and megakaryocytic cells. However, our recent work has demonstrated the down-regulation of this factor during megakaryocytic differentiation. In this study we identify two new gene targets of Gfi1b, the cytoskeletal proteins Kindlin3 and Talin1, and demonstrate the inverse expression and functions of these cytoskeletal targets relative to Gfi1b, during megakaryocytic differentiation. Both <i>kindlin3</i> and <i>talin1</i> promoters exhibit dose dependent Gfi1b and LSD1 (lysine specific demethylase 1; a Gfi1b cofactor) enrichment in megakaryocytes and repression in non-hematopoietic cells. Accordingly the expression of these genes is elevated in <i>gfi1b</i> mutant and LSD1 inhibited hematopoietic cells, while during megakaryocytic differentiation, declining Gfi1b levels fostered the reciprocal upregulation of these cytoskeletal factors. Concordantly, manipulation of Kindlin3 and Talin1 expression demonstrated positive correlation with megakaryocytic differentiation with over-expression stimulating, and inhibition diminishing, this process. Co-operativity between these factors and integrins in promoting differentiation was further underscored by physical interactions between them and integrinβ3/CD61 and by stimulation of differentiation by the Talin1 head domain, which is necessary and sufficient for integrin activation. Therefore this study demonstrates the significance of Gfi1b regulated Kindlin3-Talin1 expression in driving megakaryocytic differentiation and highlights the contribution of cytoskeletal agents in the developmental progression of these platelet progenitors.</p></div

Effect of manipulating Talin1 and Kindlin3 expression in hematopoietic progenitors.

<p>Expression of the megakaryocytic cell surface markers CD9 and CD41 in c-kit+lin- hematopoietic progenitors isolated from e13.5 fetal liver cells and cultured in vitro following transduction with the indicated shRNAs and cDNAs. Results depict one of two independent experiments.</p

Effect of manipulating Kindlin3 expression on megakaryocytic differentiation.

<p>A. Kindlin3, Talin1 and megakaryocytic marker message levels in control (Scr; scrambled) and Kindlin3 knocked down fetal liver cells differentiated into megakaryocytes. B. Acetyl choline esterase staining of the corresponding cells. P values ranged from <0.01 to <0.001 for positive cell counts. C. Message levels of indicated factors in control (pCDH; empty vector) and Kindlin3 over-expressing fetal liver cells differentiated into megakaryocytes. P values are represented by various (*, §, +) symbols for different data series with values of <0.05, 0.01 and 0.001 being indicated by one, two and three symbols, respectively. D. Acetyl choline esterase staining of the corresponding cells. The mean ± s.d. of acetylcholine-positive cells as a percentage of the total population from three independent experiments is indicated. P value was determined to be <0.05.</p