MOESM1 of Mammalian expression vectors for metabolic biotinylation tandem affinity tagging by co-expression in cis of a mammalian codon-optimized BirA biotin ligase

Additional file 1: Figure S1. Restriction maps of plasmid Avi-TEV-3xFLAG_pBS SK (A) and of plasmid 3xFLAG-TEV-Avi_pBS KS (B)

Correlation of GATA1 occupancy with absolute RP expression levels in distinct stages of mouse and human terminal erythropoiesis.

<p>(A) The absolute mRNA expression levels of RP genes in morphologically distinct stages of mouse terminal erythroid differentiation are shown. GATA1 occupancy in early (Ter119-) and late (Ter119+) erythroid differentiation stages and PU.1 occupancy in mEsEPs cells is also included. (B) The quantification of absolute mRNA levels between GATA1 occupied (red) and non-GATA1 occupied (grey) RP genes consistently shows a significant association of GATA1 binding with higher expressed RP genes (red), despite the overall decline in RP gene expression with erythroid differentiation (grey). (C) The absolute mRNA expression levels of RPs in morphologically distinct stages of human terminal erythroid differentiation and GATA1 occupancy in fetal and adult derived human erythroid cells is shown. (D) The quantification of absolute mRNA levels between GATA1 occupied (red) and non occupied (grey) RPs again shows a consistently significant association of GATA1 binding with higher expressed RP genes (red), despite the overall decline in RP gene expression with erythroid differentiation (grey).</p

GATA1 and PU.1 Bind to Ribosomal Protein Genes in Erythroid Cells: Implications for Ribosomopathies

<div><p>The clear connection between ribosome biogenesis dysfunction and specific hematopoiesis-related disorders prompted us to examine the role of critical lineage-specific transcription factors in the transcriptional regulation of ribosomal protein (RP) genes during terminal erythroid differentiation. By applying EMSA and ChIP methodologies in mouse erythroleukemia cells we show that GATA1 and PU.1 bind in vitro and in vivo the proximal promoter region of the RPS19 gene which is frequently mutated in Diamond-Blackfan Anemia. Moreover, ChIPseq data analysis also demonstrates that several RP genes are enriched as potential GATA1 and PU.1 gene targets in mouse and human erythroid cells, with GATA1 binding showing an association with higher ribosomal protein gene expression levels during terminal erythroid differentiation in human and mouse. Our results suggest that RP gene expression and hence balanced ribosome biosynthesis may be specifically and selectively regulated by lineage specific transcription factors during hematopoiesis, a finding which may be clinically relevant to ribosomopathies.</p></div

ChIP assays of GATA1 and PU.1 binding to the RPS19 proximal promoter region upon MEL cell differentiation.

<p>(A) GATA1 ChIP and (B) PU.1 ChIP of the RPS19 promoter (RPS19 prom) in MEL cells induced to terminally differentiate by treatment with 5mM HMBA. Controls include HS1 in the mouse GATA1 gene locus (GATA1+) and a negative control DNA region (GATA1-) that does not bind GATA1 based on MEL ChIPseq data. PU1+ and PU1- controls include a positive control region (PU.1+) corresponding to the Upstream Regulatory Element (URE) of the PU.1 gene locus and a negative control DNA region (PU.1-) which does not bind PU.1 on the basis of MEL ChIPseq data. No antibody ChIP controls are also shown. (C) Time course of fold-enrichment for GATA1 and PU.1 occupancies of the RPS19 promoter with MEL cell differentiation. Enrichment values for the negative control DNA regions for GATA1 (GATA1- ChIP) and PU.1 (PU1- ChIP) binding are also shown.</p

GATA1 and PU.1 ChIPseq occupancies or RP genes in erythroid cells and macrophages.

<p>GATA1 occupancies of large and small subunit RP genes (ChIPseq read density profiles, ±1.5kb around TSS is plotted) in (A) primary mouse fetal liver derived proerythroblasts (negative for Ter119 staining) and mature erythroid cells (positive for Ter119 staining)[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140077#pone.0140077.ref031" target="_blank">31</a>] and (B) in human fetal and adult erythroblasts [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140077#pone.0140077.ref043" target="_blank">43</a>]. Note that for comparison, PU.1 occupancies of RP genes in macrophages with or without LPS stimulation are also shown in (A).</p

GATA1 and PU.1 ChIP of selected mouse homologues of DBA-associated RP genes in MEL cells.

<p>ChIP assays in proliferating (A) and HMBA-differentiated (B) MEL cells. G: ChIP primers for assessing GATA1 binding; P: ChIP primers for assessing PU.1 binding; GP: ChIP primers for assessing GATA1 and PU.1 binding. ChIP primers were designed on the basis of ChIPseq data and their sequences are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140077#pone.0140077.s006" target="_blank">S2 Table</a>.</p

Gene expression fold change profiling of RP genes during human and mouse erythropoiesis in relation to GATA1 occupancy.

<p>(A) Gene expression fold change profiling of RP genes during sequential stages of human erythroid terminal differentiation with GATA1 occupancy in fetal and adult derived erythroid cells. (B) Gene expression fold change profiling of RP genes during sequential stages of mouse erythroid terminal differentiation and GATA1 occupancy in early (Ter119-) and late (Ter119+) differentiating mouse fetal liver cells. (C) Box plots of RP gene expression fold change comparing human and mouse differential expression in sequential erythroid differentiation stages, showing an overall steep decline in RP gene expression in early stages of mouse erythroid differentiation compared to human. The different stages of human and mouse erythroid differentiation were FACS purified and subjected to RNAseq as described in An et al.[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140077#pone.0140077.ref034" target="_blank">34</a>].</p

EMSA of GATA1 and PU.1 binding to the proximal promoter region of the mouse RPS19 gene.

<p>(A) Schematic representation of the mouse RPS19 gene. The translation initiation codon (ATG) was used to designate the transcription start site (TSS) and the transcription factor (TF) binding site positions. The dashed box upstream of the TSS indicates part of the sequenced RPS19 promoter region that is presented in greater detail below. The sequences for the GP (containing both the GATA1 and PU.1 sites) and P (including only the PU.1 site) EMSA probes are underlined, with the GATA1 and PU.1 consensus binding motifs indicated by italics. RPS19 promoter ChIP primers are indicated in bold. (B) EMSA showing PU.1 binding to the RPS19 promoter region. P: EMSA probe spanning the PU.1 predicted binding motif at position -653 of the RPS19 proximal promoter region. Ps: anti-PU.1 supershifted reaction; Pcom: addition of cold competitor probe; Pm: probe with PU.1 binding site mutated. (C) EMSA showing GATA1 and PU.1 binding to the RPS19 promoter region as two distinct protein complexes. GP: probe spanning the predicted PU.1 and GATA1 binding motifs at position -709bp of the RPS19 proximal promoter region. GsP: anti-GATA supershifted reaction; GPs: anti-PU.1 supershifted reaction; GmP: probe with GATA1 binding site mutated; GPm: probe with PU.1 binding site mutated; GmPm: probe with both GATA1 and PU.1 binding sites mutated.</p

Reporter gene assays showing that specific PU.1 element/s are repressed by GATA-1 in AML-ELs.

<p>The pGL3 basic plasmid was linked to the following upstream PU.1 elements: PP = proximal promoter, -12kbE, -14kbE, and the URE (or different combinations thereof, reporter constructs are named A-E were transfected into OCI-M2 and K562 cells either with scrambled control oligo (white bars) or with GATA-1 siRNA oligos (grey bars). HeLa cells served as control. Luciferase activity is normalized to the amount of proteins in each sample. Asterisks tightly to grey bars mark significances between signals from Control oligo and siRNA GATA-1 transfection within one cell line and one type of reporter. Complete ANOVA analysis results between all relevant transfections are displayed in table in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152234#pone.0152234.s004" target="_blank">S3 Fig</a>. Marks of significance: *p < 0.05, **p < 0.01, ***p < 0.001.</p

GATA-1 Inhibits <i>PU</i>.<i>1</i> Gene via DNA and Histone H3K9 Methylation of Its Distal Enhancer in Erythroleukemia

<div><p>GATA-1 and PU.1 are two important hematopoietic transcription factors that mutually inhibit each other in progenitor cells to guide entrance into the erythroid or myeloid lineage, respectively. PU.1 controls its own expression during myelopoiesis by binding to the distal URE enhancer, whose deletion leads to acute myeloid leukemia (AML). We herein present evidence that GATA-1 binds to the <i>PU</i>.<i>1</i> gene and inhibits its expression in human AML-erythroleukemias (EL). Furthermore, GATA-1 together with DNA methyl Transferase I (DNMT1) mediate repression of the <i>PU</i>.<i>1</i> gene through the URE. Repression of the <i>PU</i>.<i>1</i> gene involves both DNA methylation at the URE and its histone H3 lysine-K9 methylation and deacetylation as well as the H3K27 methylation at additional DNA elements and the promoter. The GATA-1-mediated inhibition of <i>PU</i>.<i>1</i> gene transcription in human AML-EL mediated through the URE represents important mechanism that contributes to PU.1 downregulation and leukemogenesis that is sensitive to DNA demethylation therapy.</p></div