Figure 5.
TF network that controls granulin expression. (A) Double fluorescence in situ hybridization for grna (red) and pu.1 (green) shows colocalization of both transcripts. Nuclei are stained with DAPI (blue). Pictures were taken by confocal microscopy from the tail region of 48-hpf zebrafish embryos. Each image is a 1-µm z slice. (B) WISH for the neutrophilic marker mpx (upper panels, arrowheads) or grna (lower panels, arrowheads) after MO knockdown of Pu.1 (middle panels) or Irf8 (right panels) compared with standard MO control (left panels) at 48 hpf. Pu.1 or Irf8 knockdown abolished grna expression. Asterisks denote natural pigmentation occurring in the tail of zebrafish embryos. (C) Schematic representation of the grna gene and its 5′ enhancer locus denoting 6 putative Pu.1 BSs. Pu.1 BS1-3 (green squares) were found by searching the human PU.1 target site nucleotide matrix represented in panel D using the motif comparison tool Tomtom. Pu.1 BS5-7 (orange squares) were found searching the PU.1 target site nucleotide matrix available in ConSite (http://consite.genereg.net/cgi-bin/consite). BS positions are denoted by bracketed numbers. Positive numbering starts with +1 at the A of the grna ATG translation initiation (start) codon. Nucleotides upstream (5′) of the grna ATG translation initiation codon (start) are marked with a minus sign. E, exon. White squares with a blue line indicate grna exons from the untranslated region. Blue squares represent grna coding exons. Control primers to amplify 71 base pairs of a locus within the grna gene with no predicted Pu.1 BSs for CUT&RUN normalization are indicated with a purple square. (E) CUT&RUN experiment was performed in fresh zebrafish kidney marrows from adult AB* using a Pu.1 or control immunoglobulin G (IgG) antibody. Fold enrichment of Pu.1-associated DNA fragments was identified by qPCR using primers flanking the BSs denoted in panel C. To calculate the fold enrichment, qPCR results for each BS were normalized against spike-in DNA as described86 and control primers that amplify a locus of the grna gene lacking predicted Pu.1 BSs. This experiment was performed 3 times with similar enrichments. Panel E is a representative experiment from 3 independent biological replicates performed. The primers used are shown in supplemental Table 1.

TF network that controls granulin expression. (A) Double fluorescence in situ hybridization for grna (red) and pu.1 (green) shows colocalization of both transcripts. Nuclei are stained with DAPI (blue). Pictures were taken by confocal microscopy from the tail region of 48-hpf zebrafish embryos. Each image is a 1-µm z slice. (B) WISH for the neutrophilic marker mpx (upper panels, arrowheads) or grna (lower panels, arrowheads) after MO knockdown of Pu.1 (middle panels) or Irf8 (right panels) compared with standard MO control (left panels) at 48 hpf. Pu.1 or Irf8 knockdown abolished grna expression. Asterisks denote natural pigmentation occurring in the tail of zebrafish embryos. (C) Schematic representation of the grna gene and its 5′ enhancer locus denoting 6 putative Pu.1 BSs. Pu.1 BS1-3 (green squares) were found by searching the human PU.1 target site nucleotide matrix represented in panel D using the motif comparison tool Tomtom. Pu.1 BS5-7 (orange squares) were found searching the PU.1 target site nucleotide matrix available in ConSite (http://consite.genereg.net/cgi-bin/consite). BS positions are denoted by bracketed numbers. Positive numbering starts with +1 at the A of the grna ATG translation initiation (start) codon. Nucleotides upstream (5′) of the grna ATG translation initiation codon (start) are marked with a minus sign. E, exon. White squares with a blue line indicate grna exons from the untranslated region. Blue squares represent grna coding exons. Control primers to amplify 71 base pairs of a locus within the grna gene with no predicted Pu.1 BSs for CUT&RUN normalization are indicated with a purple square. (E) CUT&RUN experiment was performed in fresh zebrafish kidney marrows from adult AB* using a Pu.1 or control immunoglobulin G (IgG) antibody. Fold enrichment of Pu.1-associated DNA fragments was identified by qPCR using primers flanking the BSs denoted in panel C. To calculate the fold enrichment, qPCR results for each BS were normalized against spike-in DNA as described86  and control primers that amplify a locus of the grna gene lacking predicted Pu.1 BSs. This experiment was performed 3 times with similar enrichments. Panel E is a representative experiment from 3 independent biological replicates performed. The primers used are shown in supplemental Table 1.

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