Biochimica et Biophysica Acta 1839 (2014) 50–61

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Biochimica et Biophysica Acta

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Cohesin and CTCF differentially regulate spatiotemporal runx1expression during zebrafish development

Judith Marsman 1, Adam C. O'Neill 1, Betty Rui-Yun Kao 1, Jenny M. Rhodes, Michael Meier, Jisha Antony,Maren Mönnich, Julia A. Horsfield ⁎Department of Pathology, Dunedin School of Medicine, The University of Otago, P.O. Box 913, Dunedin, New Zealand

Abbreviations: CRE, cis-regulatory element; CTCF, CCCpolymerase II; PLM, posterior lateral mesoderm; ALM, aRohon–Beard neurons; ICM, intermediate cell mass; MOTSS, transcription start site⁎ Corresponding author. Tel.: +64 3 479 7436; fax: +6

E-mail address: julia.horsfield@otago.ac.nz (J.A. Horsfi1 These authors contributed equally to this work.

1874-9399/$ – see front matter © 2013 Elsevier B.V. All rhttp://dx.doi.org/10.1016/j.bbagrm.2013.11.007

a b s t r a c t

a r t i c l e i n f o

Article history:Received 30 July 2013Received in revised form 19 November 2013Accepted 25 November 2013Available online 7 December 2013

Keywords:Runx1Rad21CohesinCTCFZebrafishHematopoiesis

Runx1 is a transcription factor essential for definitive hematopoiesis. In all vertebrates, the Runx1 gene is tran-scribed from two promoters: a proximal promoter (P2), and a distal promoter (P1). We previously found thatrunx1 expression in a specific hematopoietic cell population in zebrafish embryos depends on cohesin. Herewe show that zebrafish runx1 is directly bound by cohesin and CCCTC binding factor (CTCF) at the P1 and P2 pro-moters, and within the intron between P1 and P2. Cohesin initiates expression of runx1 in the posterior lateralmesoderm and influences promoter use, while CTCF represses its expression in the newly emerging cells ofthe tail bud. The intronic binding sites for cohesin and CTCF coincide with histone modifications that conferenhancer-like properties, and two of the cohesin/CTCF sites behaved as insulators in an in vivo assay. The identi-fied cohesin andCTCF binding sites are likely to be cis-regulatory elements (CREs) for runx1 since they also recruitRNA polymerase II (RNAPII). CTCF depletion excluded RNAPII from two intronic CREs but not the promoters ofrunx1. We propose that cohesin and CTCF have distinct functions in the regulation of runx1 during zebrafish em-bryogenesis, and that these regulatory functions are likely to involve runx1 intronic CREs. Cohesin (but not CTCF)depletion enhanced RUNX1 expression in a human leukemia cell line, suggesting conservation of RUNX1 regula-tion through evolution.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Runx1 is a transcription factor essential for regulating the develop-ment and maintenance of definitive hematopoietic stem cells duringembryogenesis. Embryonic disruption of the Runx1 gene is lethal inmid-gestation due to complete failure of definitive hematopoiesis ac-companied by extensive hemorrhaging [1,2]. In contrast, conditionalablation of Runx1 in adult bone marrow only has mild effects on hema-topoiesis [3,4], suggesting that at some point during hematopoietic de-velopment Runx1 activity is no longer required [5–7]. In the nervoussystem, Runx1 also has a role in the development of dorsal root gangliaand cranial sensory neurons [8–10], highlighting its potential to regu-late the development of multiple cell lineages.

Runx1 belongs to the runt-domain containing “Runx” family of tran-scription factors. All vertebrates have three Runx genes, each of whichhas crucial roles in the differentiation of specific cell lineages fromstem cells [11,12]. All Runx genes have two promoters, proximal anddistal, which are used to transcribe distinct isoforms of the genes

TC-binding factor; RNAPII, RNAnterior lateral mesoderm; RB,, morpholino oligonucleotide;

4 3 479 7136.eld).

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(Fig. 1). The dual promoter structure of vertebrate Runx genes, con-served over 250 million years of evolution [13], is likely to be importantfor their correct regulation. Vertebrate Runx1 is transcribed from a prox-imal promoter (P2), and a distal promoter (P1) to generate transcriptswith alternative 5′UTRs and first coding exons [14–16]. In mammals,transcription of Runx1 from its two promoters generates up to three iso-forms of the protein: Runx1a and 1b from P2, and Runx1c from P1[17,18]. The diverse Runx1 isoforms add complexity to Runx1 expres-sion and function [7,19,20], and could mediate differential control ofcell-type and -stage specific developmental programs. Because ofRunx1's important developmental role and its involvement in cancer[21], particularly leukemia [22], there has been intense interest in theelucidation of factors that regulate its expression.

In early zebrafish embryos, runx1 transcripts first appear in hemato-poietic precursors of the posterior lateral mesoderm (PLM) at the4-somite stage, and in primitive myeloid cells of the anterior lateralmesoderm (ALM) at 8 somites [23,24]. By around 18 hours post-fertilization (hpf), cells in the PLM have migrated medially to form acentral rod of hematopoietic precursors: the intermediate cell mass(ICM). After that stage, hematopoietic expression of runx1 is downreg-ulated in all but the most posterior cells of the ICM, and later reappearsin definitive hematopoietic precursors in the ventral wall of the dorsalaorta at around 24 hpf [23]. runx1 is also expressed in Rohon–Beard(RB) mechanosensory neurons and in the olfactory placode [23].In human hematopoietic cell lines [25] and in mouse [26–28],

Fig. 1.Comparative genomic organization of Runx1 loci in human,mouse and zebrafish re-veals conservation of the P1 5′UTR. (A) Genomic organization of the human, mouse andzebrafish runx1 locus. The exons are numbered according to the UCSC Genome browserand those that are part of the 3′ and 5′ untranslated regions (UTRs) are shown as whiteboxes. The splice variants runx1a and runx1b are transcribed from the proximal P2 pro-moter and the splice variant runx1c from the distal P1 promoter. Distances between thepromoters are indicated in kilobases (kb). (B) Schematic overview and sequence of the5′ end of the zebrafish runx1c isoform. The UTRs (light gray), coding regions (dark gray),transcriptional start site (TSS) and translational start site (white) are indicated. Locationsof the sequence are listed according to the UCSC Genome browser.

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cis-regulatory elements (CREs) in the intron between P1 and P2 arethought to mediate cell type-specific Runx1 regulation. In contrast,hematopoietic expression of runx1 in zebrafish is not thought to requireCREs, with the promoters themselves conferring hematopoietic activity[29].

Previously, we determined that cell type-specific expression ofrunx1 in the PLM from 6 to 16 hpf depends on the chromatin proteincomplex cohesin [24]. Cohesin consists of four core subunits: Smc3,Smc1, Rad21 and SA1/SA2; it is best known for its role in sister chroma-tid cohesion [30], but has important emerging roles in DNA damage re-pair and gene transcription [31,32]. Zebrafish embryos null for cohesinsubunit Rad21, or depleted of cohesin subunit Smc3, entirely lack PLMexpression of runx1 but retain expression in RB neurons and still havesome ALM expression [24]. Altered runx1 gene expression occurred inembryos that had sufficient maternally deposited cohesin subunitRad21 for further cell divisions. This suggested that there is a thresholdlevel of cohesin below which cell proliferation can be sustained, but atwhich gene expression in selected cells is compromised [24]. However,the mechanism for this extraordinary cell type-specific nature of runx1regulation by cohesin remained unknown.

Cohesin and CCCTC-binding factor (CTCF), have emerged as keyregulators of isoform-specific and tissue-specific gene regulation(reviewed in [33]). The cohesin complex preferentially binds transcrip-tionally active genes, where it interacts with the basal transcriptionma-chinery and chromatin modifiers, likely with functional consequencesfor gene activation (reviewed in [31]). CTCF is best known for its insula-tor function, where it demarcates chromatin domains and restricts theinfluence of enhancers on selected gene loci [34,35]. CTCF recruitscohesin to chromosome arms [33–39] and so these proteins are widelybelieved to cooperate in tissue-specific gene regulation. One potentialmechanism for this regulation is the positioning of distant regulatoryelements into proximity with gene promoters [33,39]. There is also ev-idence that cohesin and CTCF regulate alternative promoter usage. Forexample, cohesin and CTCF control isoform-specific expression of theclustered protocadherin genes in mouse brain, via chromatin interac-tions that select which promoter isoform will be transcribed [40–42].

Here we sought to identify the mechanism by which cohesin regu-lates zebrafish runx1. We found multiple predicted and in vivo bindingsites for cohesin and CTCF in the zebrafish runx1 gene, including atboth promoters and within the intron between them. Binding sites forcohesin coincided with putative CREs in the zebrafish runx1 intron be-tween P1 and P2. These CREs contain active chromatin and associatedwith RNA polymerase II (RNAPII) in the presence of cohesin and CTCF.Cohesin binds both promoters of runx1, and since its depletion is associ-atedwith loss of RNAPII, positive runx1 regulation by cohesin is likely tobe direct. In contrast, CTCF appears to restrict the expression pattern ofrunx1, consistent with insulator activity. We propose that cohesin andCTCF have distinct functions in the regulation of runx1 during zebrafishembryogenesis, and that these regulatory functions are likely to involvethe runx1 intronic CREs. Binding sites for cohesin and CTCF are con-served in human and mouse Runx1; furthermore, cohesin depletionincreased expression of RUNX1 in a human cell line, suggesting evolu-tionary conservation of RUNX1 regulation.

2. Material and methods

2.1. Zebrafish lines

Zebrafish were maintained as described previously [43]. TheUniversity of Otago Animal Ethics Committee approved all zebrafishresearch.

2.2. 5′ RACE

Total RNA was extracted using Trizol (Invitrogen) from pools of80–150 embryos at 24–33 hpf, and purified using Qiagen RNeasy col-umns. The FirstChoice RLM-RACE Kit (Ambion) was used to amplifythe 5′ untranslated regions (UTRs) of runx1-P1 and runx1-P2. Primersused for RACE are listed in Supplementary Table S1.

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2.3. Microinjection

Morpholino oligonucleotides (MOs) were obtained from GeneToolsLLC and diluted in water with Phenol Red. MO sequences are given inSupplementary Table S1. For microinjection, 1 nl containing 0.5 pmolof each MO was injected into the yolk of wild type embryos at the 1–2cell stage. Characterization of the rad21 and ctcfMOswas described pre-viously [44]. Microinjection of ctcf MO for quantitative PCR was doneusing 1 nl containing 0.25 pmol splice-blocking MO (ctcfSplx2-MO)combined with 0.25 pmol translation-blocking MO (ctcfATG-MO). Ef-fectiveness of MOs in targeting protein production was confirmed byWestern blot (Fig. S1).

2.4. Cell culture and transfection

Human myelocytic leukemia cells (HL-60) were maintained at adensity of 1–10 × 105 cells/ml in RPMI-1640 (R8755 Sigma Aldrich)supplemented with 4.5 g/l glucose (Sigma Aldrich), 5.96 g/l HEPES(Acros Organics), 1 mM NaPyruvate (Invitrogen), 2 g/l NaHCO3

(Sigma Aldrich) and 10% FBS (Moregate, New Zealand). HL-60 cellswere provided by A. Braithwaite (University of Otago, Dunedin, NewZealand). Cells were incubated in a 37 °C humidified incubator at 5%CO2. Passage 4–9 cells were transfected with 50 or 100 nM siRNAusing the Neon® Transfection System (Invitrogen) and collected 48 hpost-transfection. 5 × 105 cells were used per 100 μl transfection reac-tion and cells were seeded at a density of 5 × 105 cells/ml. The siRNAsused (Thermo Scientific), previously characterized in [45], were: RAD21siRNA J006832-06, Ctrl siRNA D-001810-01, CTCF siRNA SMARTpool L-020165-00, Ctrl siRNA SMARTpool D-001810-10.

2.5. Quantitative RT-PCR

Total RNA from pools of 30–80 embryos or ~1 × 106 HL-60 cells wasextracted using NucleoSpin® RNA II Kit (Machery-Nagel). 500 ng(human) or 1 μg (zebrafish) RNA was used to synthesize random-primed cDNA (SuperScriptIII, Invitrogen). Platinum® SYBR® GreenqPCR SuperMix-UDG with ROX (Invitrogen) was used to amplifycDNA; per 20 μl reaction, 2 μl of undiluted zebrafish cDNA, 1 μl of undi-luted human cDNA for RUNX1-P1, 1 μl of 1 in 5 diluted human cDNAfor RUNX1-P2, RUNX1-total, and housekeeping genes was used. Cpvalues were determined by the 2nd derivative method using RocheLightCycler® 480 software. In zebrafish, the Pfaffl method [46] wasused to calculate transcript expression relative to the average Cp valueof all samples. PCR amplification efficiencies were determined basedon the average of three biological replicates of a standard curve. Differ-ences in input cDNAbetween sampleswere normalized to cDNAsampleconcentrations using the average of three Nanodrop 1000 measure-ments. In human, transcript expression was normalized to the house-keeping genes GAPDH, RPL13A and CYCLOPHILIN using qBase Plus(Biogazelle) and results are represented as fold change in expressionlevel relative to control sample (mean ± SEM, n = 3). Graphs and sta-tistical analyses were produced using Prism (GraphPad Software). PCRprimers are listed in Table S1.

2.6. Whole-mount in situ hybridization

Full length runx1 riboprobe synthesis and whole mount in situhybridization was performed as described previously [23]. A templatefor the runx1-P1 riboprobewas amplified using the forward and reverseprimers shown in Table S1, and the template for runx1-P2was amplifiedusing the same primers as used for RACE. Fragments were cloned intopGEM-T-easy (Promega). The runx1-P1 plasmid was linearized withApaI and the runx1-P2 with NcoI; antisense DIG-labeled riboprobeswere generated by in vitro transcription using SP6 RNA polymerase(Roche).

2.7. Imaging

Embryos were imaged using a LeicaM205FA stereomicroscope witha DFC490 camera and LAS software (Leica Microsystems).

2.8. Database identification of cohesin and CTCF binding sites

Genomic runx1 sequences of the zebrafish assembly zv9/danRer7,mouse assembly NCBI37/mm9 and human assembly GRCh37/hg19were obtained from the UCSC Genome Browser (http://genome.ucsc.edu/) under accession numbers NM_131603, NM_001111021 andNM_001122607 respectively. Genomic DNA sequences of Runx1 usedin this study correspond to locations chr1:1048741–1221029 inzebrafish, chr16:92598711–92849311 in mouse and chr21:36150098–36431595 in human. Promoter and CRE sequences in zebrafish, mouseand human Runx1 were acquired from the literature [14,28,29]. CTCFbinding sites in zebrafish were predicted using an online tool athttp://insulatordb.utmem.edu/ [47]. Binding sites were annotatedusing Geneious software (Biomatters).

2.9. Enhancer blocking assay

An insulator test (INS) vector [48] was used to investigate theenhancer-blocking capacity of the four runx1 CREs found in this study.This vector contains a cardiac actin promoter that drives GFP expressionin the somites and an upstream Z48 enhancer that drives GFP expres-sion in themidbrain [48].When an insulator element is placed betweenthe enhancer and promoter, midbrain GFP expression is repressed. DNAsequences for CREs were amplified, cloned into pCR®8/GW/TOPO entryvector and recombined into the INS vector using the Gateway® system(LR Clonase® Enzyme mix, Invitrogen). Primers used to amplify thecloned regions can be found in Table S1. 1–2 nl of 30 ng/μl vector DNAand 30 ng/μl Tol2 transposasemRNA [49]was injected into 1–2 cell em-bryos. At least 10 individual embryos were imaged from two indepen-dent experiments at 36 h post-fertilization (hpf). GFP intensity in thesomite and midbrain areas was quantified using Image J. Graphs andstatistical analyses were produced using Prism (GraphPad Software).

2.10. Antibodies

Antibodies used for ChIP and Western Blot assays were: anti-Rad21[44] (raised in rabbit against a 15 amino acid peptide of the zebrafish pro-tein, GenScript Corporation, USA), anti-H3K4Me1 (ab8895; Abcam), anti-H3K27Ac (ab4729; Abcam), anti-pan histone H3 (05-928; Millipore),anti-RNA polymerase II (ab5131; Abcam), anti-CTCF (raised in rabbitagainst a 15 amino acid peptide of the zebrafish protein, GenScript Corpo-ration, USA), anti-RAD21 (ab992; Abcam), anti-CTCF (CTE3417S; CellSignaling) and mouse anti-γ-tubulin (Sigma Aldrich). Specificity of cus-tom antibodies detecting zebrafish Rad21 and zebrafish CTCF was con-firmed by Western blot (Fig. S1).

2.11. Chromatin immunoprecipitation (ChIP)

ChIP was performed as described previously [44,50] on chromatinprepared from pools (n = 750–1500) of 6, 14 and 24 hpf embryos.Chromatin was sheered to an average size of 300–600 base pairs (bp)such that ChIP peaks could be resolved to within a kilobase. Sampleswere analyzed by quantitative PCR using SYBR Green (Life Technolo-gies) and an Applied Biosystems 7300 Real-Time PCR System. Graphsand statistical analyses were produced using Prism (GraphPad Soft-ware). All PCR primers are listed in Table S1.

2.12. Western blotting

Cells were lysed in Radio-Immunoprecipitation Assay (RIPA) buffercontaining protease inhibitors (Complete™, Roche). Zebrafish embryos

Fig. 2. Rad21 and CTCF depletion have differing effects on runx1 transcript distribution inearly zebrafish embryos. Whole mount in situ hybridization with a full-length runx1riboprobe was performed on wild type, rad21 morphant and ctcf morphant embryos at10–12 somites. A, C, F, H are posterior views while B, D, G and I are lateral views with an-terior to the left. E, J are pictorials of dorsal, flattened views of embryos. (A, B) wild typeexpression pattern of runx1, with PLM expression indicated by arrows. (C, D) runx1expression in rad21morphant embryos. Expression in the stripes of the PLM is eliminated(arrows) while trace expression is retained in the ALM (asterisk). Expression in the RBneurons is unaffected. (E) diagram summarizing zones of runx1 expression in 8–14 somiteembryos. Blue zones indicate expression that is insensitive, or only partially sensitive toRad21 depletion. Green zones indicate abolished runx1 expression in rad21 mutants ormorphants. OP, olfactory placode; ALM, anterior lateral mesoderm; RB, Rohon-Beard neu-rons; PLM, posterior lateral mesoderm. (F, G) runx1 expression in ctcfmorphants. Ectopicrunx1 expression in the tail bud is indicated by red arrowheads. (H, I) runx1 expression inrad21/ctcf double morphants (co-injection of 0.5 pmol of each morpholino oligonucleo-tide). Note the absence of PLM expression but retention of runx1 expression in RB cellsand in the ALM. (J) diagram summarizing effects of Rad21 and CTCF depletion on runx1expression in 8–14 somite embryos. Green zones, cells that require cohesin for activationof runx1 expression. Red zone, cells that require CTCF for repression of runx1 expression.Blue zones, cells in which runx1 expression is independent of cohesin and CTCF.

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were dechorionated and deyolked prior to lysis. Protein concentrationwas measured using Bicinchoninic Acid (BCA) assay (Pierce, Thermo-scientific) and equal amounts of protein were separated by electropho-resis on 10% polyacrylamide gels. Proteins were transferred toNitrocellulose membranes (Thermoscientific) and blocked using theOdyssey® blocking buffer and Odyssey® Casein blocking buffer(LiCor), according to the manufacturer's instructions. The membraneswere incubated with anti-RAD21 (1:1000 for human and 1:500 forzebrafish), anti-CTCF (1:1000) and mouse anti-γ-tubulin (1:5000,Sigma) diluted in Odyssey® blocking buffer and Odyssey® Caseinblocking buffer for 3 h at room temperature. After washing with PBS-Tween, the membranes were incubated with anti-mouse IRDye® 680(1:15,000, LiCor #926-32220) and anti-rabbit IRDye® 800 (1:15,000LiCor #926-32211) for one hour at room temperature, and visualizedwith the Odyssey® CLx Infrared imaging system (LiCor). Band intensi-ties were quantified using Image J.

3. Results

3.1. Cohesin and CTCF differentially affect tissue-specific transcription andpromoter use of the runx1 gene

We previously found that cohesin regulates spatiotemporal runx1transcription in hematopoietic precursors of the PLM and the olfactoryplacode from 6 to 16 hpf [24]. Since cohesin-dependent runx1 regula-tion is restricted to specific cells, we were curious to know if transcrip-tion from one or both runx1 promoters is cohesin-dependent. Inaddition, previous studies have shown that CTCF recruits cohesin tochromosomes [51], and can cooperatewith cohesin to regulate gene ex-pression [31]. Thus it is possible that CTCF, like cohesin, regulates runx1.Therefore, we investigated the quantity and distribution of P1- and P2-driven transcripts in early zebrafish embryos thatwere either wild type,rad21mutant, or depleted for Rad21 and CTCF using splice-blocking ortranslation-blocking antisense morpholino oligonucleotides (MO).

We used 5′RACE to identify the TSS and unique 5′ sequences forzebrafish P1 (runx1c) and P2 (runx1a/b) transcripts (Figs. 1, S2). Ampli-fication of unique 5′ sequence for the runx1c isoform revealed an intronin the non-coding 5′UTR (Fig. 1A,B). Genomicmapping of 174 bp 5′UTRsequence indicated that the TSS for runx1c is 265 bp upstream of theATG (Fig. 1B), providing new transcript information for the zebrafishrunx1 gene. The TSS of runx1 transcribed from P2 was mapped to288 bp upstream of the start of the first exon following P2 (Fig. S2).These data are consistent with evolutionary conservation of the struc-ture of Runx1 transcripts between zebrafish and mammals (Fig. 1A).

To analyze the embryonic expression pattern of runx1 transcripts, 5′UTR-specific riboprobes for whole mount in situ hybridization weregenerated, each of which incorporated the 5′UTR and unique first trans-lated exon of runx1-P2 or runx1-P1. However, we could not detect thescarce transcript runx1-P1 by in situ hybridization with the short174 bp P1 probe. At 10–12 somites, expression of runx1-P2 recapitulat-ed the entire expression pattern of runx1 (Figs. 2A, B; S3). As deter-mined previously [24], rad21nz171 mutant embryos completely lackedexpression of runx1 in the PLM (Fig. 2A–E). Since runx1-P2 is expressedin the PLM, its expression there must be cohesin dependent. However,runx1-P2 distribution is not restricted to PLM cells: it is also expressedin RB cells in which runx1 expression does not require cohesin(Fig. S3). Since runx1-P2 is expressed in both a cohesin-dependentand cohesin-independent manner, the effects of cohesin on runx1 tran-scription must be restricted to a specific cell type: cells of the PLM.

We knocked down the CTCF protein (ctcfSplx2-MO, ctcfATG-MO) aspreviously described [44], to produce CTCFmorphants (Fig. S1). Surpris-ingly, CTCF knockdown did not eliminate runx1 expression from thePLM, but instead, induced ectopic runx1 expression in newly emergingcells of the tail bud (Figs. 2F, G; S4). This suggests that CTCF normally re-presses runx1 expression in these cells (Fig. 2J), and is not required foractivation of runx1 in the PLM. Furthermore the ectopic expression

generated by CTCF depletion appears to be cohesin-dependent. Doubleknockdown of CTCF and cohesin (rad21ATG-MO) gave an identical phe-notype to the knockdown of cohesin alone: no PLM expression of runx1,together with no tail bud expression (Fig. 2H, I). Therefore CTCF sup-presses runx1 expression in undifferentiated tail bud cells while cohesinindependently activates its expression in the PLM (Fig. 2E, J).

To determine if cohesin and CTCF can differentially affect transcrip-tion from the promoters of runx1, we knocked down the expression ofthese proteins in zebrafish embryos using MOs as previously described[44], and used quantitative PCR tomeasure P1- and P2-generated runx1transcripts. Depletion of Rad21 decreased expression of runx1 overall in13-somite embryos (Fig. 3A), but surprisingly caused 2–3-fold increasein the relative levels of the P1 transcript at 13 somites and at 24 hpf(Fig. 3B). In contrast, CTCF depletion had little effect on overall runx1

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Fig. 3.Rad21 and CTCFdepletion havediffering effects on runx1promoter use. Expression of runx1 transcribed from the P1 or P2 promoterswas determined byquantitative RT-PCR inwildtype (WT), Rad21-depleted and CTCF-depleted embryos, at 13 somites and 24 h post fertilization (hpf). The graphs represent relative expression of the isoforms compared to the averageCp value of all samples (see Material and methods). (A) The average of at least three biological replicates ± SEM is shown as a stacked bar graph. Differences in mean P1 expression at24 hpf betweenWT, Rad21-depleted and CTCF-depleted embryos was statistically significant (p b 0.01; one-way ANOVA). The asterisk indicates p b 0.01 (Dunnett's multiple compari-sons test). (B) Expression driven by P1 and P2 shown as a percentage stacked bar, representing relative expression from either promoter.

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expression at 13 somites, and did not influence promoter choice(Fig. 3A, B). By 24 hpf, total runx1 levels were not significantly affectedby Rad21 or CTCF depletion (Fig. 3A). Our results indicate that Rad21/cohesin is required for early runx1 transcription and can influencerunx1 promoter usage, while CTCF is not essential for runx1 transcrip-tion from either promoter.

3.2. CTCF and cohesin directly bind the runx1 locus

To determine if cohesin and CTCF can directly regulate runx1 tran-scription, we asked if these proteins bind to the runx1 promoters or toother regulatory sequences. No genome-wide binding data is yet avail-able for cohesin and CTCF in zebrafish, sowe used locations of potential-ly conserved binding sites in human and mouse to predict putativeCTCF/cohesin binding sites in zebrafish runx1. In addition, we used aCTCF binding site prediction tool [47] to predict binding sites from up-stream of P1 to the start of P2. As well as the promoters and transcrip-tion start sites (TSS), the inter-promoter intron was chosen for thisanalysis because regulatory elements exist there in mammals [36,37].

We assayed binding to the predicted zebrafish sites using ChIP ofchromatin from 13-somite and 24 hpf zebrafish embryos with antibod-ies detecting Rad21 and CTCF (Figs. 4; S1). There was robust cohesin(Fig. 4B, D) and CTCF (Fig. 4C, E) binding to predicted sites located at+14 and +89 downstream of the runx1-P1 TSS at both embryonicstages. Usingmore primer sets to obtain a closer dissection of the region,we were able to detect Rad21 binding to another predicted location at+39 and the TSS of both runx1 isoforms at the 13-somite stage(Fig. 4F). Binding of cohesin and CTCF to the runx1 locus indicatespotential for these factors to directly regulate its transcription.

3.3. Cohesin and CTCF binding coincide with runx1 intronic CREs

Sites co-occupied by cohesin and CTCF can either have an activatingor inhibiting function, whereas sites bound by cohesin in the absence ofCTCF are often tissue-specific regulatory elements [33]. The mouseRunx1 locuswas previously found to contain CREs in the formof two en-hancers, at+24 and+25 kb downstreamof the Runx1c TSS [26,27], be-tween the two Runx1 promoters. The +24 enhancer sequence isconserved from frog to human (but appears to be absent in zebrafish),while the +25 enhancer is conserved in mammals only [27]. We won-dered if the binding sites for cohesin and CTCF overlap with potentialCREs between P1 and P2. Because DNA sequence between zebrafishand mouse is poorly conserved in this region [29], we used ChIP inzebrafish embryos with antibodies detecting chromatin marks that

characterize enhancers [52–54] to survey locations around predictedand confirmed cohesin and CTCF binding sites.

There was amarked enrichment of histone H3mono-methylated onlysine 4 (H3K4Me1, marks regulatory elements) at +13 and +39 kbdownstream from the runx1-P1 TSS in 6 hpf, 13-somite and 24 hpfzebrafish embryos (Fig. 5). Enrichment of H3K4Me1 was not due tolocal changes in histone H3 distribution (Fig. S5), suggesting that CREsare likely present at these locations. Atmost stages of zebrafish develop-ment surveyed, both CREs were enrichedwith histone H3 acetylated onlysine 27 (H3K27Ac), a mark of active chromatin (Fig. 5B–D). The localenrichment of H3K27Ac relative to total levels of histoneH3 (Fig. S5) in-dicates that enrichment likely represents active chromatin rather thanaltered histone H3 distribution.

Significantly, the +13 and +39 CREs are close to cohesin and CTCFbinding sites at +14 and +39 kb (Fig. 4). The proximity of bindingsites for cohesin and CTCF to putative CREs indicates that these elementshave potential to influence runx1 transcription. The+89 binding site forcohesin and CTCF is within the P2 promoter [29], and its proximity tothe transcription start site (b2 kb) indicates that this regulatory ele-ment is also likely to be in active chromatin and influence transcription.

3.4. Some CREs bound by CTCF and cohesin can function as insulators

CTCF is well known for its insulator activity [33]. Since the CREs at+14, +39 and +89 recruit cohesin and CTCF, it is possible that theyhave insulator or enhancer function. Therefore, we used an insulatorand enhancer zebrafish assay system [48] to determinewhether the pu-tative CREs have transcriptional activity in vivo.

To detect insulator activity, we used a vector (INS) containing thezebrafish irx Z48 enhancer driving GFP expression in the midbrain,with a cardiac actin promoter driving GFP in somites and heart [48]. Ac-tive insulator sequences are predicted to decrease irx Z48-mediatedEGFP expression in themidbrain, without changing somite GFP expres-sion. The cardiac actin-driven GFP expression in the somites serves inthis assay as an internal control for GFP normalization. Putative CREswere amplified, cloned into the INS vector and injected with mRNAencoding Tol2 transposase into zebrafish embryos at the 1-cell stage.At 36 hpf, EGFP expression in midbrain and somites was quantitatedin the embryos (Fig. 6A) and relative values graphed (Fig. 6B). GFP ex-pression was repressed in the midbrain relative to the somites in em-bryos with INS harboring the +13, +14 and +89 CRE sequences(Fig. 6A, B). By contrast, the+39 sequence did not appear to have insu-lator activity in this assay (Fig. 6B). To determine if the +13, +14 and+89 sequences depend on CTCF for their insulator activity, we depletedCTCF using a combination of ctcf Splx2-MO and ctcfATG-MO in embryos

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Fig. 4. Rad21 and CTCF bind to multiple locations at the start of the runx1 gene. (A) Schematic of the zebrafish runx1 gene. Arrows indicate the location of the runx1-P1 and runx1-P2transcription start sites (TSS). Numbers represent the distance from the runx1-P1 TSS in kb and indicate the locations that were tested for Rad21 and CTCF binding. Fold enrichment ofboth Rad21 and CTCF is relative to a negative control site at +12 (not bound by either protein). (B) Rad21 binding in 24 hpf wild type embryos showed significant enrichment at +14and+89 (near the runx1-P2 TSS), with a suggestion of binding at+39. (C) CTCF binding in 24 hpf wild type embryos also showed significant enrichment at +14 and+89, with modestbinding at +39. (D, E) Rad21 and CTCF binding in 13-somite wild type embryos was enriched at +14 and +89. (F) Assessment of additional locations in 13-somite wild type embryosconfirmbinding of Rad21 to−0.1 (runx1-P1 TSS),+14,+39,+89 (runx1-P2 promoter) and+91 (runx1-P2 TSS). For B–F, error bars represent ± SEMof at least two biological replicatesat each location tested. Changes in enrichment of Rad21 or CTCF at each time point were statistically significant (p b 0.01; one-way ANOVA). Asterisks indicate statistically significantdifferences compared to the lowest data point of each data set (*p b 0.05, **p b 0.01; Dunnett's multiple comparisons test).

55J. Marsman et al. / Biochimica et Biophysica Acta 1839 (2014) 50–61

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Fig. 5. Enrichment of H3K4Me1 and H3K27Ac in the zebrafish runx1 locus identifiescis-regulatory elements. (A) Schematic representation of the runx1 locus indicating theprimer positions (horizontal bars) used to amplify immunoprecipitated DNA followingChIP. Primer positions are indicated in kb from the transcriptional start site of runx1-P1.(B–D) Enrichment of anti-H3K4Me1 (filled circles, solid line) and H3K27Ac (open circles,dotted line) at indicated positions across the runx1 locus in chromatin from 6 hpf (B),13-somite (C) and 24 hpf (D) embryos. Enrichment at each site is expressed as a percent-age of the input. Results shown are the average of two independent ChIP experiments ±SEM.Changes in enrichment of H3K4me1 andH3K27ac at each timepointwere statistical-ly significant (p b 0.0001; one-way ANOVA). Asterisks indicate statistically significantdifferences compared to the lowest data point of each data set (* p ≤ 0.0001; Dunnett'smultiple comparisons test). Raw data used to produce the line graphs in B–D can befound in Fig. S5.

56 J. Marsman et al. / Biochimica et Biophysica Acta 1839 (2014) 50–61

injected with the INS harboring these putative CREs (Fig. 6C).We foundthat partial reduction of CTCF protein diminished the ability of all CREsto repress midbrain EGFP expression (statistically significant for the+89 CRE) (Fig. 6D). The results suggest that the +13, +14 and +89CREs are competent to act as insulators in a CTCF-dependent manner.

To determine if the +39 element instead had enhancer activity, weused the Tol2 transposon-based Zebrafish Enhancer Detector (ZED)vector, which contains pgata2 as a minimal promoter [48] and the car-diac actin promoter driving RFP. In RFP-positive 36 hpf embryos, wecould not detect any transient GFP expression driven by the +39 CREvia the pgata2 minimal promoter (data not shown). Strong enhancers

have been detected using transient GFP expression in this system [48]therefore it is unlikely that the +39 CRE has strong enhancer activity.However, we cannot rule out that this CRE has weak enhancer activity.

It is possible that the CREs we identified (Fig. 5) recruit other tran-scriptional regulators. Analyses using the transcription element searchsystem tool, TESS [55], predict binding sites for hematopoietic transcrip-tion factors including Runx1, Gata1, Ets, Myb, CEBP and E-box bindingfactors in all CRE sequences (data not shown). Together with detectedinsulator activity for +13, +14 and +89, our evidence indicates thatthe sites bound by cohesin and CTCF are likely to have potential to influ-ence the transcription of runx1.

3.5. CTCF recruits cohesin and RNAPII to CREs of runx1

CTCF was previously found to recruit cohesin to chromosomes [33].To determine if CTCF recruits cohesin to zebrafish runx1, we knockeddown CTCF and performed anti-Rad21 ChIP in 13-somite embryos.CTCF was necessary to recruit cohesin to its intronic binding sites atthe runx1 gene. Strong reduction of Rad21 binding was observed atthe +14 and +39 sites (70%), while a much more modest reductionof Rad21 binding was detected at the +89 binding site (30%) and TSS(40–50%) of both promoters (−0.1, +91) in ctcfmorphants comparedwith wild type (Fig. 7A, B). This could indicate that cohesin binding atselected sites has differential dependency on CTCF, and/or that cohesinbinds to different sites in cells that express runx1 versus cells that donot.

In ctcf morphants, the runx1 locus must be transcriptionally active,since runx1 mRNA is present (Fig. 3) and ectopically expressed in cellsof the tail bud of 13-somite embryos (Fig. 2F, G). In addition, twoCTCF-bound CREs (+13/14, +89) had insulator activity in an in vivoassay (Fig. 6). We therefore asked whether the cohesin/CTCF-boundCREs are involved in transcription by conducting ChIP with antibodiesdetecting RNAPII. Significantly, ChIP of chromatin from ctcf morphantsrevealed that RNAPII was diminished at the +13 and +39 CREs uponCTCF depletion (Fig. 7C). However, RNAPII remained bound to the TSSof both runx1 promoters. RNAPII was not significantly depleted fromthe promoter-proximal site +89, which also retains a significant pro-portion of Rad21 binding (Fig. 7B). By contrast, when Rad21was deplet-ed, RNAPII wasmarkedly reduced at the TSS of both promoters of runx1,consistent with transcription downregulation (Fig. S6).

Our results indicate that in CTCF-depleted embryos, transcriptionalactivity no longer involves the +13/14 and +39 CREs, since RNAPII isdisplaced from these elements upon CTCF knockdown. Therefore, it ispossible that ectopic runx1 expression in the tail bud of CTCFmorphants(Fig. 3F, G) arises from loss of insulator function of the +13 and +14elements (Fig. 6). Cohesin is necessary for ectopic expression of runx1in CTCFmorphants (Fig. 3H, I), and retention of Rad21 and RNAPII bind-ing at site +89 in CTCF morphants raises the possibility that this partic-ular CRE is crucial for cohesin-dependent runx1 transcription.

3.6. Cohesin depletion enhances RUNX1 transcription in human cells

Genome-wide binding data from ENCODE indicate that cohesin andCTCF also bind the RUNX1 locus in mouse and human. Therefore, it ispossible that cohesin and CTCF could regulate RUNX1 expression inmammals, including humans. In K562 cells (a human chronic myeloidleukemia cell line), cohesin and CTCF locate to both RUNX1 promotersand several sites in intron 2, including at the +24/+25 CRE identifiedby Ng et al. [27] (Fig. 8A). To determine if cohesin and CTCF can regulateRUNX1 transcription in vivo, we diminished these proteins in HL-60human myelocytic leukemia cells using siRNA (Fig. S7). Strikingly, wefound that cohesin (but not CTCF) depletion resulted in a marked(N50%) increase in transcription from both promoters of RUNX1,with increased transcription from P1 being statistically significant(p b 0.05). The results are consistent with those observed for 24 hpfRad21- and CTCF-depleted zebrafish embryos (Fig. 3), and indicate

WT CTCFCTCF

g-tubulin

A B

C

D

Fig. 6. runx1 cis-regulatory elements block enhancer activity in vivo. (A) Representative images of 36 hpf embryos injected at the 1–2 cell stagewith an empty INS vector (control), lackingan insert between the enhancer and promoter, or an INS vector containing the core region of the runx1+13,+14, +39 and+89 cis-regulatory elements. Expression in themidbrain (ar-rows) becomes repressed relative to somite expression if an insulator sequence is present. (B) Box–whisker plot showing the somite/midbrain relative GFP expression (quantified usingImage J). A Kruskal–Wallis test resulted in a significant difference (p b 0.0001) andwas followed by a Dunn's multiple comparisons test. Asterisks indicate the set of values that were sig-nificantly different from the control; *: p = 0.001 and **: p b 0.0001. (C)Western blot analysis of CTCF and γ-tubulin protein inwild type (WT) versus CTCF-depleted embryos at 36 hpf,using protein extracted from embryos analyzed in D. Quantification showed ~33% reduction of CTCF protein in CTCF-depleted embryos. (D) Box–whisker plot showing the somite/mid-brain relativeGFP expression (quantified using Image J) in embryos injectedwith INS vectors containing the runx1 CREs as shown in B, compared to embryos injected simultaneouslywithCTCF splx2 and ATGmorpholinos. Somite/midbrain relativeGFP expression in CTCF-depleted embryos injectedwith the+89 elementwas statistically significant from embryos containingwild type CTCF protein levels (*p b 0.05; Dunn's multiple comparisons test).

57J. Marsman et al. / Biochimica et Biophysica Acta 1839 (2014) 50–61

that a role for cohesin in Runx1 regulation is likely to be conservedthrough evolution.

4. Discussion

4.1. Cohesin and CTCF have distinct roles in the control of cell type-specificrunx1 expression

Our previous work showed that of all the transcription factors wesurveyed, runx1 alonewas absent in PLM cells [24]. Several other hema-topoietic transcription factors, including Pu.1, Scl, and the dimerizationpartner of Runx1, Cbf, are expressed normally in the PLMof cohesinmu-tants [24]. Interestingly, runx1 expression is retained in the RB neurons,and although reduced, runx1 is still expressed in the ALMof cohesinmu-tants (Fig. 2D). Therefore, among transcriptional regulators surveyed,runx1 alone is exquisitely sensitive to cohesin levels, within a tight de-velopmental window, and with cohesin-dependent expression restrict-ed to the PLM hematopoietic cells [34].

Since runx1 has two promoters that can drive transcription of differ-ent isoforms, we wondered if cohesin or CTCF depletion affects tran-scription from just one of the promoters. runx1-P2 was expressed inall runx1-expressing tissues (Fig. S3), so we concluded that this isoformis not overall differentially regulated, but rather, only sensitive tocohesin levels in PLM cells. In rad21morphants at 13 somites, the totalamount of runx1 transcript was decreased overall (Fig. 3A), consistentwith its disappearance from the PLM. The decrease is likely to bedue to downregulated transcription from the P2 promoter, which

contributes to the bulk of runx1 transcription at this stage (Fig. 3A).Interestingly, relative quantities of the rare transcript from the P1 pro-moter increased when Rad21 was depleted, at both 13 somites and24 hpf (Fig. 3B). This could indicate that cohesin contributes to a pro-moter isoform switching mechanism for runx1, or that its activity is re-quired tomaintain transcription from P2 in particular. In support of thisinterpretation, promoter isoform selection by cohesin was also ob-served for mouse protocadherin loci [51].

Since CTCF and cohesin are thought to act in the same direction toregulate gene expression [51], we expected CTCF depletion to abolishexpression of runx1 in the PLM. Surprisingly, CTCF-depleted embryosretained the normal expression pattern of runx1, and gained additionalexpression in newly emerging cells of the tail bud (Figs. 2F, G; S4). Thedata indicate that cohesin and CTCF need not necessarily have identicalfunctions in the regulation of gene expression, even at regulatory loca-tions where CTCF promotes cohesin binding. Our data add to a growinglist of examples of distinct roles for cohesin and CTCF [44,56].

4.2. Evidence for direct regulation of runx1 by cohesin and CTCF

Direct regulation of runx1 by cohesin and CTCF is supported by evi-dence that they directly bind the locus at numerous sites, includingthe runx1 promoters, and other elements located at +13, +39 and+89 downstream of the P1 TSS (Fig. 4). Significantly, sites bound byCTCF at +13, +14 and +89 had CTCF-dependent insulator activity inan in vivo zebrafish assay (Fig. 6). A predicted ability to recruit tissue-specific transcription factors and an in vivo insulator function for some

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Fig. 7. Depletion of CTCF alters Rad21 and RNAPII binding to the runx1 locus. (A) Schematic representation of the runx1 locus indicating the primer positions (horizontal bars) used toamplify immunoprecipitated DNA following ChIP. Primer positions are indicated in kb from the transcriptional start site of runx1-P1. Intronic CRE locations (+13, +39) are in italics,additional Rad21 binding sites are at+14 and+89, and primers amplifying the TSS of runx1-P1 are at -0.1; runx1-P2 at+91. B and C represent ChIP of chromatin from 13-somite embryosat the indicated positions in A. Fold enrichment of both Rad21 and RNAPII is relative to a negative control site at +12 (not shown). (B) Anti-Rad21 ChIP in wild type (gray bars) and ctcfmorphants (black bars). Enrichment of Rad21 was markedly reduced at the +14 and +39 CREs, and partially reduced at the +89 CRE and P2 promoter of runx1 in ctcfmorphants com-pared to wild type, while enrichment at the control sites (−0.7, +37, +38, +40, +90) was unaffected. (C) Anti-RNAPII ChIP in wild type (gray bars) and ctcfmorphants (black bars). Enrich-ment of RNAPII was reduced at the+13 and+39 CREs of runx1 upon CTCF depletion and partially reduced at+90 kb, while enrichment at the TSSs (−0.1,+91) and control sites (−0.7,+37,+38, +40) was unaffected. Results shown are representative of at least two biological replicates ± S.E.M. * p b 0.05 (Student's T-test).

58 J. Marsman et al. / Biochimica et Biophysica Acta 1839 (2014) 50–61

of the runx1 CREs signifies that they can likely restrict the spatial expres-sion of runx1. The +89 site is less than 2 kb from the runx1-P2 TSS andcould therefore be considered to be part of the P2 promoter. Its insulatoractivity suggests that it may act to restrict transcription from P2.

It should be noted that the ChIP experiments described here havethe limitation thatwe analyzed chromatin fromwhole zebrafish embry-os, which represent a mixed cell population. It is possible that cohesinand CTCF bind different sites when runx1 is transcribed compared towhen runx1 is inactive. However, transcriptional activity of CREs andpromoter sites was cohesin- and CTCF-dependent. RNAPII binding wasreduced at the runx1 promoters in rad21 morphants, consistent withdownregulation of runx1 (Fig. S6). In ctcf morphants, RNAPII was re-duced at the +13 and +39 CREs but substantially retained at P2 andthe +89 CRE (Fig. 7), consistent with maintenance of runx1 transcrip-tion. Therefore, it is likely that cohesin/CTCF binding at the CREs is ac-tively involved in runx1 expression.

Collectively, our results support the hypothesis that ectopic runx1expression in CTCF-depleted embryos occurs because the +13/14 and+39 CREs can no longer interact with the runx1 promoters, resultingin loss of CTCF-mediated insulator activity that restrains runx1 expres-sion in the tail bud. We cannot rule out an alternative interpretation,which is that CTCF depletion results in inappropriate differentiation ormigration of stem cells in the tail bud, in turn resulting in ectopicrunx1 expression.

4.3. Conservation of runx1 gene structure among vertebrates

At the amino acid sequence level, Runx1 orthologs are well con-served among species [13] reflecting their important function in devel-opmental processes. Furthermore, the genomic structure of runx1 ishighly conserved among vertebrates [13]. New information fromthe present study shows that, in addition to conservation of the2-promoter structure, the intron in the 5′UTR of the runx1c isoform isconserved between mammals and zebrafish. Conservation of the 5′UTR intron raises the possibility that it is important for the regulationof Runx1 expression.

In mouse, at least two enhancers exist, and are found +24 and+25 kb downstream of the TSS of Runx1c [26,27]. Our study raises thepossibility that similar regulatory elements may be conserved inzebrafish. When we investigated the nature of histone modificationspresent at cohesin and CTCF binding sites located at +13 and +39 kbdownstream of the runx1-P1 TSS, we found that these binding siteshad enhancer-like hallmarks (Figs. 5; S5). Analysis with TESS softwareindicated that these sites have potential to recruit hematopoietic tran-scription factors (data not shown). However, the +13, +14 and +89binding sites were also capable of acting as insulators (Fig. 6), and wehave not yet been able to detect enhancer activity driven by these ele-ments, at least in transient assays. The roles of the CREs in zebrafishare unclear, but it seems likely that they have the potential to control

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Fig. 8. Binding and regulation of Runx1 by cohesin is conserved between zebrafish and human. (A) Experimentally identified binding sites of cohesin (based on Rad21 and/or Smc3binding; brown) and CTCF (pink) are indicated at the human and zebrafish Runx1 loci. In human, experimentally identified binding sites in K562 cells were obtained from ChIP-seqdata of ENCODE/Yale, ENCODE/Stanford, ENCODE/broad, ENCODE/HudsonAlpha, ENCODE/UW and ENCODE/UT-A. Locations of cis-regulatory elements (CRE; green) in human wereobtained from Ng. et al. (ref #27). In zebrafish, the in vivo binding sites identified in this study are shown. Base pair numbers, P1 and P2 promoters (hooked arrows) and exons (gray)are indicated. (B) Relative expression of the RUNX1 P1 isoform, P2 isoform and total RUNX1 in HL-60 cells transfected with control (Ctrl; single siRNA), RAD21 (single siRNA), controlfor CTCF (pool of four siRNAs) and CTCF (pool of four siRNAs). Quantitative PCR data was normalized to the housekeeping genes GAPDH, RPL13A and CYCLOPHILIN, and expressed relativeto the corresponding control. The average of three biological replicates ± SEM is shown (*p b 0.05, **p b 0.01; Student's T-test).

59J. Marsman et al. / Biochimica et Biophysica Acta 1839 (2014) 50–61

spatiotemporal runx1 expression in the early zebrafish embryo. An ear-lier study showed that when isolated from their native context, therunx1 promoters are able to faithfully drive GFP expression in hemato-poietic cells from around 18 hpf [29]. However, prior to 18 hpf thesites of P1- and P2-driven GFP expression do not appear to accuratelyreflect in situ hybridization data for runx1 expression (Fig. S8,[23,24,29]). The data are consistentwith additional regulatory elementsbeing required for the correct initiation of runx1 expression in zebrafishembryos, with later runx1 expression in definitive hematopoietic cellsbeing independent of the CREs.

4.4. A conserved role for cohesin and CTCF in Runx1 transcription?

Our present study provides new insight into roles of cohesin andCTCF in the initiation of runx1 expression in the developing embryo.Cohesin and CTCF are known to mediate long-range interactions be-tween CREs and promoters [33]. Our data is consistent with a mecha-nism in which cohesin and CTCF regulation of runx1 involves contactbetween the CREswe identified and the runx1 promoters. Chromosomeconformation capture (3C) experiments on single cell types would berequired to confirm this hypothesis.

The presence of cohesin/CTCF binding sites in human Runx1 raisesthe possibility that these proteins control Runx1 expression in mam-mals. In support of this idea, we observed that depletion of RAD21(but not CTCF) enhanced RUNX1 transcription in human HL-60 myelo-cytic leukemia cells (Fig. 8), similar to our observations in 24 hpfzebrafish embryos. In addition, mRNA levels of RAD21 and RUNX1were found to be dysregulated and co-dependent in endometrial cancer[57]. Intriguingly, recent analyses have shown that up to 13% of acutemyeloid leukemia (AML) samples carry mutations in cohesin subunitsor cohesin regulators [58,59]. AML is frequently characterized by alteredRUNX1 function [22]. Yoshida et al. [60] demonstrated a striking

association of cohesin mutation with myeloid dysplasia. Down's syn-drome (DS) individuals can present with transient abnormalmyelopoiesis (TAM) that is self-limiting inmost cases. TAM is amyeloidproliferation resembling AML, and 10% of TAM progresses to non self-limiting acute megakaryoblastic leukemia (AMKL) in DS (DS-AMLK).Deep sequencing revealed that 53% of DS-AMLK samples had acquiredcohesin mutations that were not found in somatic cells or the originalTAM [60]. This finding is strongly suggestive of a causative relationshipbetween cohesin mutation and progression to AMLK, whichmay possi-bly be linkedwith the extra copy of RUNX1 in DS. Future studies will de-termine if cohesin and CTCF have a role in leukemia via RUNX1regulation, or if they mediate developmental transcription of Runx1 inother species.

5. Conclusions

Wehave shown that cohesin and CTCF have distinct functions in theregulation of runx1during zebrafish embryogenesis. Regulation of runx1by cohesin and CTCF is likely to be direct, and mediated through associ-ations with newly identified CREs at the runx1 locus. A hypotheticalmodel for runx1 regulation by cohesin and CTCF is proposed in Fig. 9.Binding sites for cohesin and CTCF are present in mammalian genes,and RUNX1 transcription was altered in a RAD21-depleted human he-matopoietic cell line (Fig. 8). Therefore cohesin- and CTCF-dependentregulation of Runx1 has potential to be conserved in mammals.

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.bbagrm.2013.11.007.

Acknowledgement

We thank Motomi Osato for assistance with TESS predictions andhelpful discussions, and we are grateful to Phil Crosier for supplying

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Fig. 9.Hypothetical model for runx1 gene configuration in different cell types of the zebrafish embryo. The schematics in A and B represent hypothetical chromosome configurations thatcould exist in different cell types in the zebrafish embryo, contributing to an ability to drive cell type-specific expression of runx1. (A) Posterior mesoderm cells, which depend on cohesinand CTCF for the correct spatial pattern of runx1 expression,may contain a clustered configuration of the runx1 gene promoters and inter-promoter intron sequences. In this configuration,CREs are brought into contact with the two promoters (blue rectangles), contain active chromatin, and are bound by a transcription complex containing RNAPII. The action of CTCF andassociated insulator sequences (red rectangles) restrains runx1 expression in newly emergent, undifferentiated cells of the tailbud. Formation of the hypothetical transcription complexmay depend on cohesin since it is necessary for runx1 expression in the PLM. (B) In the absence of CTCF, the+13/+14 and+39 CREs no longer bind RNAPII, implying they are no longer inthe complex. However, the promoters and the +89 CRE still bind cohesin and RNAPII, possibly forming a cohesin-dependent secondary structure that facilitates positive regulation ofrunx1. (C) In cells where runx1 expression is not dependent on cohesin, e.g., RB cells, secondary structures do not form at all, and runx1 regulation is under the control of promoter-specific transcription factors. Similarly, in rad21 mutants or morphants, all cohesin-dependent secondary structures are lost, and PLM cells are no longer able to activate runx1.

60 J. Marsman et al. / Biochimica et Biophysica Acta 1839 (2014) 50–61

the Tg(runx1P2:EGFP) line, José Bessa for providing the INS and ZED vec-tors and Antony Braithwaite for providing HL-60 cells. We thank NoelJhinku for expertmanagement of theOtago Zebrafish Facility, and JustinO'Sullivan for a critical reading of the manuscript. This research wassupported by the Royal Society of New Zealand Marsden Fund (11-UOO-027, 07-UOO-037 to J.H.) and Lottery Health Research NZ (LHR-278199 to J.H.).

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