Optimal use of tandem biotin and V5 tags in ChIP assays

Background: Chromatin immunoprecipitation (ChIP) assays coupled to genome arrays (Chip-on-chip) or massive parallel sequencing (ChIP-seq) lead to the genome wide identification of binding sites of chromatin associated proteins. However, the highly variable quality of antibodies and the availability of epitopes in crosslinked chromatin can compromise genomic ChIP outcomes. Epitope tags have often been used as more reliable alternatives. In addition, we have employed protein in vivo biotinylation tagging as a very high affinity alternative to antibodies. In this paper we describe the optimization of biotinylation tagging for ChIP and its coupling to a known epitope tag in providing a reliable and efficient alternative to antibodies. Results: Using the biotin tagged erythroid transcription factor GATA-1 as example, we describe several optimization steps for the application of the high affinity biotin streptavidin system in ChIP. We find that the omission of SDS during sonication, the use of fish skin gelatin as blocking agent and choice of streptavidin beads can lead to significantly improved ChIP enrichments and lower background compared to antibodies. We also show that the V5 epitope tag performs equally well under the conditions worked out for streptavidin ChIP and that it may suffer less from the effects of formaldehyde crosslinking. Conclusion: The combined use of the very high affinity biotin tag with the less sensitive to crosslinking V5 tag provides for a flexible ChIP platform with potential implications in ChIP sequencing outcomes

Optimal use of tandem biotin and V5 tags in ChIP assays

Background: Chromatin immunoprecipitation (ChIP) assays coupled to genome arrays (Chip-on-chip) or massive parallel sequencing (ChIP-seq) lead to the genome wide identification of binding sites of chromatin associated proteins. However, the highly variable quality of antibodies and the availability of epitopes in crosslinked chromatin can compromise genomic ChIP outcomes. Epitope tags have often been used as more reliable alternatives. In addition, we have employed protein in vivo biotinylation tagging as a very high affinity alternative to antibodies. In this paper we describe the optimization of biotinylation tagging for ChIP and its coupling to a known epitope tag in providing a reliable and efficient alternative to antibodies. Results: Using the biotin tagged erythroid transcription factor GATA-1 as example, we describe several optimization steps for the application of the high affinity biotin streptavidin system in ChIP. We find that the omission of SDS during sonication, the use of fish skin gelatin as blocking agent and choice of streptavidin beads can lead to significantly improved ChIP enrichments and lower background compared to antibodies. We also show that the V5 epitope tag performs equally well under the conditions worked out for streptavidin ChIP and that it may suffer less from the effects of formaldehyde crosslinking. Conclusion: The combined use of the very high affinity biotin tag with the less sensitive to crosslinking V5 tag provides for a flexible ChIP platform with potential implications in ChIP sequencing outcomes

Biased, Non-equivalent Gene-Proximal and -Distal Binding Motifs of Orphan Nuclear Receptor TR4 in Primary Human Erythroid Cells

<div><p>We previously reported that TR2 and TR4 orphan nuclear receptors bind to direct repeat (DR) elements in the ε- and γ-globin promoters, and act as molecular anchors for the recruitment of epigenetic corepressors of the multifaceted DRED complex, thereby leading to ε- and γ-globin transcriptional repression during definitive erythropoiesis. Other than the ε- and γ-globin and the <i>GATA1</i> genes, TR4-regulated target genes in human erythroid cells remain unknown. Here, we identified TR4 binding sites genome-wide using chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq) as human primary CD34⁺ hematopoietic progenitors differentiated progressively to late erythroid precursors. We also performed whole transcriptome analyses by RNA-seq to identify TR4 downstream targets after lentiviral-mediated TR4 shRNA knockdown in erythroid cells. Analyses from combined ChIP-seq and RNA-seq datasets indicate that DR1 motifs are more prevalent in the proximal promoters of TR4 direct target genes, which are involved in basic biological functions (e.g., mRNA processing, ribosomal assembly, RNA splicing and primary metabolic processes). In contrast, other non-DR1 repeat motifs (DR4, ER6 and IR1) are more prevalent at gene-distal TR4 binding sites. Of these, approximately 50% are also marked with epigenetic chromatin signatures (such as P300, H3K27ac, H3K4me1 and H3K27me3) associated with enhancer function. Thus, we hypothesize that TR4 regulates gene transcription <i>via</i> gene-proximal DR1 sites as TR4/TR2 heterodimers, while it can associate with novel nuclear receptor partners (such as RXR) to bind to distant non-DR1 consensus sites. In summary, this study reveals that the TR4 regulatory network is far more complex than previously appreciated and that TR4 regulates basic, essential biological processes during the terminal differentiation of human erythroid cells.</p></div

Number and percentage of epigenetic enhancer marks that co-localize with TR4 distal peaks.^*

<p>*Within ±2 Kbp of TR4 distal peaks.</p

Sequence conservation among TR4 peaks.

<p>(<b>A</b>) The percentage of peaks located >10 Kbp from the nearest RefSeq genes decreased from D8 to D14 of differentiation. (<b>B</b>) For peaks located at the proximal promoter, the average PhastCons score of each nucleotide within a peak (500 bp from peak center) across vertebrate, mammalian or primate species are graphed. The center of each peak is defined as “0”. (<b>C</b>) Comparison of the average PhastCons scores of peak sequences and random control sequences in the TR4 peaks located at proximal promoter, >10 Kbp from genes or in all identified peaks at D14 differentiation.</p

Distribution of potential NR binding sites in TR4-bound peaks.

<p>Peak sequences (250 bp from peak center) were interrogated using NHR-scan for the presence of direct repeat (DR), everted repeat (ER) and inverted repeat (IR) motifs with 0–8 spacer nucleotides in D8, D11 and D14 (<b>A</b>, <b>B</b>) or in D8 (<b>C</b>) cells. The percentages of each motif type in peaks located at the proximal promoter (<b>A</b>), in all peaks (<b>B</b>), and in peaks at proximal promoter <i>vs</i>. >10 Kbp from genes (<b>C</b>) are represented graphically.</p

Characterization of TR4 downstream targets with lentiviral-mediated shRNAs.

<p>(<b>A</b>) The upper panel shows the immunoblots of TR4 and β-actin (internal control) in shRNA lentivirus (#174 and #658) or control virus infected cells at D11. The bar graph shows the relative abundance of TR4 normalized to β-actin and to control cells (*<i>p</i><0.05 and error bars represent s.e.m.). (<b>B</b>) Venn diagram summarizing the common and unique differentially expressed genes after TR4 depletion with lentivirus sh#174 and sh#658. (<b>C</b>) The enriched GO terms for TR4 repressed and activated genes, respectively.</p

The expression of genes with TR4 bound at proximal promoter is reduced after TR4 depletion.

<p>(<b>A</b>) The distribution of the expression fold change between genes with TR4 bound in the proximal promoter region (dashed red lines) <i>vs</i>. that of all expressed genes (solid black line) after TR4 depletion by either lentivirus sh#174 (right panel) or lentivirus sh#658 (left panel), where positive values indicate an increase in expression after TR4 depletion. (<b>B</b>) Genes with TR4 bound at the proximal promoter are enriched in basic biological functions.</p

Total raw reads and peaks from TR4 ChIP-seq analyses of differentiating human erythroid cells.

<p>Total raw reads and peaks from TR4 ChIP-seq analyses of differentiating human erythroid cells.</p