Drosophila melanogaster dHCF Interacts with both PcG and TrxG Epigenetic Regulators

Repression and activation of gene transcription involves multiprotein complexes that modify chromatin structure. The integration of these complexes at regulatory sites can be assisted by co-factors that link them to DNA-bound transcriptional regulators. In humans, one such co-factor is the herpes simplex virus host-cell factor 1 (HCF-1), which is implicated in both activation and repression of transcription. We show here that disruption of the gene encoding the Drosophila melanogaster homolog of HCF-1, dHCF, leads to a pleiotropic phenotype involving lethality, sterility, small size, apoptosis, and morphological defects. In Drosophila, repressed and activated transcriptional states of cell fate-determining genes are maintained throughout development by Polycomb Group (PcG) and Trithorax Group (TrxG) genes, respectively. dHCF mutant flies display morphological phenotypes typical of TrxG mutants and dHCF interacts genetically with both PcG and TrxG genes. Thus, dHCF inactivation enhances the mutant phenotypes of the Pc PcG as well as brm and mor TrxG genes, suggesting that dHCF possesses Enhancer of TrxG and PcG (ETP) properties. Additionally, dHCF interacts with the previously established ETP gene skd. These pleiotropic phenotypes are consistent with broad roles for dHCF in both activation and repression of transcription during fly development

ChIP analysis of the <i>Mkrn3</i> and <i>Ndn</i> promoters.

<p>A) ChIP analysis of the <i>Mkrn3</i> locus. Antibodies against NRF-2, Sp1, and YY1 were used to immunoprecipitate chromatin from the maternal and paternal alleles separately in Tg^PWSdel and Tg^ASdel mouse fibroblasts, respectively. The location of primers used to examine transcription factor binding within regions 1–4 across the <i>Mkrn3</i> locus are described further in the main text. The solid rectangle depicts the intronless <i>Mkrn3</i> gene; the bent arrow represents the transcription initiation site. Open bars represent analysis of the maternal allele, bars with horizontal stripes represent control samples from Tg^PWSdel cells (maternal allele) treated with no antibody, solid bars represent analysis of the paternal allele, and bars with vertical stripes represent control samples from Tg^ASdel cells (paternal allele) treated with no antibody. B) ChIP analysis of the <i>Mkrn3</i> promoter region (region 2 in panel A) in primary mouse brain and spleen cells. Brain and spleen cell preparations from C57BL/6 mice were subjected to ChIP analysis with antibodies against YY1, NRF-2, or RNA polymerase II. C) ChIP analysis of the <i>Ndn</i> promoter region in Tg^PWSdel and Tg^ASdel cells using antibodies against NRF-1, YY1, and Sp1.</p

Mapping of DNase I hypersensitive sites in the <i>Mkrn3</i> locus.

<p>Intact cells were treated with increasing concentrations of DNase I and purified genomic DNA was analyzed by indirect end-labeling by Southern blotting as described in Materials and Methods. Maternal and paternal alleles were analyzed separately using Tg^PWSdel (PWS) and Tg^ASdel (AS) fibroblasts. Lanes marked “0” represent samples of purified mouse genomic DNA from Tg^PWSdel or Tg^ASdel cells. “Probe” indicates location of hybridization probe used in Southern blot analysis.</p

<i>In vivo</i> footprint analysis of the <i>Snrpn</i> promoter region.

<p>LMPCR <i>in vivo</i> footprinting with dimethyl sulfate (DMS) was performed on primary brain cells isolated from Tg^PWS(del) and Tg^AS(del) mice carrying a deletion of the entire murine AS/PWS region on either the paternal or maternal chromosomes, respectively <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052390#pone.0052390-Gabriel1" target="_blank">[20]</a>. Cells were treated with increasing time of exposure to DMS. “DNA” represents control samples of purified genomic DNA subjected to DMS treatment and LMPCR in parallel with samples treated <i>in vivo</i> with increasing time of exposure to DMS (60, 90, 120 seconds). The LMPCR primer sets detecting each footprint are described in the text. A) – D) Autoradiograms showing <i>in vivo</i> footprints P1 – P5 in the <i>Snrpn</i> promoter region. Filled circles represent footprints detected as guanines protected from DMS reactivity, open circles represent footprints at guanine sites showing enhanced DMS reactivity. Numbers on the left of each sequencing gel denote the location of each footprint relative to the transcription initiation site. The nucleotide sequences to the right of each autoradiogram show the DNA sequence containing and flanking each footprint; the filled/open circles indicate the location of each <i>in vivo</i> footprint within the DNA sequence and correspond to the footprinted sites shown on the left of each autoradiogram. E) Summary of <i>in vivo</i> footprints in the <i>Snprn</i> promoter region of the paternal allele. The footprinted sites detected by LMPCR <i>in vivo</i> footprinting are shown within the nucleotide sequence of the promoter region of the mouse <i>Snprn</i> gene. Open and filled circles denote footprinted sites as described above. At each footprinted position, the footprinted site corresponds to the strand containing the guanine nucleotide. Shaded nucleotide sequences indicate sequences conserved between the human and mouse <i>Snrpn</i> promoters. Brackets below the nucleotide sequence indicate the location and identity of potential transcription factor binding sites. The bent arrow depicts the transcription initiation site; numbering of nucleotides is relative to the transcription initiation site.</p

Chromosome conformation capture (3C) analysis of the <i>Mkrn3</i> locus.

<p>A) Diagram of 3C analysis of the <i>Mkrn3</i> locus showing the location of the anchor primer and primers <i>a</i>, <i>d</i>, and <i>e</i>. Bent arrows indicate transcription initiation sites. Short horizontal bars depict the location of primers <i>a</i>, <i>d</i>, <i>e</i>, and the anchor primer. Short vertical marks below the magnified <i>Mkrn3</i> and <i>Snrpn</i> 5′ regions indicate the location of EcoRI sites. The regions surrounding <i>Mkrn3</i> and the <i>Snrpn</i> transcription initiation site are shown approximately to scale. The long horizontal brackets show the approximate distance between the <i>Snrpn</i> and <i>Mkrn3</i> promoter regions, and the relative location of the 35 kb PWS-IC deletion in the <i>Snrpn</i> locus <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052390#pone.0052390-Yang1" target="_blank">[18]</a>. B) 3C analysis of Tg^PWSdel and Tg^ASdel fibroblasts using primers <i>a</i> and <i>d</i> with the anchor primer. The figure shows an ethidium bromide-stained agarose gel containing the products of PCR reactions between the anchor primer and the indicated primer. <i>Mat</i> indicates analysis of the maternal allele in Tg^PWSdel cells, <i>Pat</i> indicates analysis of the paternal allele in Tg^ASdel cells. “<b>−</b>” indicates a non-ligated control template, “<b>+</b>” indicates the ligated template, and “W” indicates a control PCR reaction with an equal volume of H₂O substituted for a 3C template. C) 3C analysis of primary mouse brain cells. 3C analysis was performed on single-cell suspensions of newborn brains from mice carrying the 35 kb PWS-IC deletion on either the maternal or paternal chromosome. All designations are the same as those previously described. Primers <i>a</i>, <i>d</i> and the anchor primer were identical to those used in panel B. Primer <i>e</i> is located within the same EcoRI fragment as primer <i>a</i>. <i>Pat</i> denotes 3C templates from brain cells carrying the 35 kb PWS-IC deletion on the maternal chromosome, <i>Mat</i> denotes 3C templates containing the PWS-IC deletion on the paternal chromosome. “G” indicates control purified mouse genomic DNA.</p

ChIP analysis of transcription factor binding to the <i>Snrpn</i> locus.

<p>Chromatin immunoprecipitation analysis was performed to assay for binding of NRF-1 and YY1 to DH sites in the <i>Snrpn</i> 5′ region in primary brain and spleen cells of C57Bl/6 mice. A) Diagram of the mouse <i>Snrpn</i> 5′ region showing the regions assayed by ChIP. Pairs of opposing horizontal arrows depict the location of PCR primer sets used for real-time PCR in ChIP assays. Primer sets 1 and 3 are negative controls and amplify regions where no known factors are thought to be bound. B) Results of ChIP assays performed with antibodies against the transcription factors NRF-1 and YY1. Black and gray vertical bars denote the results for the ChIP experiments on brain cells and spleen cells of C57B/6 mice, respectively. Numbers under the graphs indicate the results obtained for the corresponding PCR primer sets shown in panel A.</p

DNase I hypersensitivity of the murine PWS-IC.

<p>DNase hypersensitive sites (DHS) were mapped by Southern blotting and indirect end-labeling after DNase I treatment of primary brain cells. The maternal and paternal alleles were analyzed separately using brain cells prepared from mice carrying a 35 kb PWS-IC deletion on either the paternal or maternal chromosome, respectively <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052390#pone.0052390-Bielinska1" target="_blank">[8]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052390#pone.0052390-Yang1" target="_blank">[18]</a>. Cells were treated with increasing concentrations of DNase I; <i>0 DNase</i> samples were purified genomic DNA from untreated brain cells. (A) Analysis by EcoRI digestion and hybridization with probe A. The thick arrows indicate the positions and sizes of prominent reproducible DNase I hypersensitive bands. <i>Paternal</i> indicates samples carrying a maternal PWS-IC deletion; <i>Maternal</i> indicates samples carrying a paternal PWS-C deletion. B) Analysis by Taq I digestion and hybridization with probe B. C) Analysis by Nco I digestion and hybridization with probe C. D) Summary of DNase I hypersensitive sites and their locations. The diagram depicts the <i>Snrpn</i> 5′ region showing relevant restriction enzyme sites and the positions of hybridization probes A, B and C (probe B is contained within probe C). The relative positions of DHS1-6 are indicated by short horizontal bars above the gene; all prominent DH sites (DHS1-DS6) are specific to the paternal chromosome. <i>CAS</i> denotes the conserved activator sequence <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052390#pone.0052390-RodriguezJato1" target="_blank">[17]</a> that co-localizes with DHS5. Thin horizontal lines below the gene depict regions deleted by targeted knockouts of the <i>Snrpn</i> locus <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052390#pone.0052390-Bressler1" target="_blank">[38]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052390#pone.0052390-Tsai1" target="_blank">[39]</a>. The bent arrow depicts the location of the transcription initiation site. The position of each DH site relative to the transcription initiation site is estimated to be: DHS1, −3.2 kb; DHS2 includes the transcription initiation site; DHS3, +0.8 kb; DHS4, +1.4 kb; DHS5, +1.7 kb; DHS6, +4.1 kb; the positions of DHS1-DHS6 in the <i>Snrpn</i> gene were calculated as an average of at least two independent experiments and/or different restriction enzymes and probes. The same DH sites mapped by different experiments, restriction enzymes, and/or blots generally localized within 200–300 bp of each other, a variation within the range expected by estimating positions derived from band sizes in Southern blots.</p