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To identify if PU.1 was able to directly bind IR5, a chromatin

immunoprecipitation (ChIP) assay was employed. ChIP is a method for assaying protein-DNA ineractions. In the assay, formaldehyde is used to crosslink protein to DNA in cells (i.e transcription factors to the DNA promoters of genes) so that any interactions (which may be transient) are stabilised throughout the experiment. The nucleii are then extracted and the genomic DNA is sheared, followed by subsequent immunoprecipitation of the transcription factor of interest and analysis of any co-immunoprecipitated DNA by PCR and/or sequencing.

For ChIP analysis, pcDNA4-PU.1 or pcDNA4-vector only (VO, as a control) were transfected into HEK 293T cells. After formaldehyde crosslinking genomic DNA was sheared by sonication to 200-500bp in length. The number of sonications had to be empirically optimised for each cell type in order to generate fragments of the correct size; too few sonications would produce larger DNA fragments whilst too many sonications would produce much smaller DNA fragments. The optimum number of sonications for the HEK 293T cells was 4 x 15s (Figure 4.5).

Figure 4.5: Optimisation of HEK 293T genomic DNA shearing for subsequent ChIP analysis. HEK 293T cells were treated with formaldehyde to crosslink protein to

DNA prior to lysis by sonication. For accurate ChIP analysis, the optimal genomic DNA fragment length is 200-500bp, which is achieved through differential amounts of sonication followed by analysis by visualisation on an ethidium bromide stained 1% agarose gel. 4 x 15s pulses of sonication produce the correct sized fragments (4 x 15s sonications), whereas less sonications (2 x 15s) produce a longer smear of genomic DNA on the gel representing larger DNA fragments due to insufficient sonication.

The pcDNA4 vector encompasses an Xpress® epitope tag onto the subcloned

DNA, and so the ectopically expressed PU.1 or the VO were immunoprecipitated with an anti-Xpress antibody. Protein immunoprecipitation was confirmed by Western blot (Figure 4.6A). As a positive control for the ChIP assay, primers for the previously characterised CD11b promoter to which PU.1 binds (Brugnoli et al., 2009) were used alongside primers to encompass IR5 of the LIMD1 promoter. When PU.1 was immunoprecipitated, both the CD11b and LIMD1

promoter regions that encompass the PU.1 binding motif were co-

immunoprecipitated, as detected by PCR (Figure 4.6). These regions were not detected when the VO was precipitated, showing that ectopic PU.1 specifically binds to the LIMD1 promoter in vivo in HEK 293T cells.

1000- 800- 600- 400- 200- bp

Figure 4.6: Exogenous PU.1 binds to the LIMD1 promoter in HEK 293T cells.

pcDNA4-PU.1 or vector only (VO) was transfected into HEK 293T cells. 48 hours post transfection cells were serum starved overnight, then stimulated with 20% FCS before protein and DNA were crosslinked and DNA was sheared by sonication. (A) PU.1 or control VO were immunoprecipitated with 2.5µg of anti-Xpress and confirmed by Western blot probed with anti-PU.1. (B) Co-immunoprecipitated DNA was purified and analysed by PCR utilising primers for the previously characterised CD11b promoter as a positive control along with primers encompassing the PU.1 motif within the LIMD1 promoter.

To then ascertain the relevance of ectopically expressed PU.1 bound to the LIMD1 promoter the interaction between endogenous PU.1 and the LIMD1 promoter was assayed. As PU.1 is expressed at high levels in haematopoietic derived cell lineages, the U937 histiocytic lymphoma cell line was assayed for

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Following immunopre spanning the PU.1 bin The CD11b promoter result was consisten expressed PU.1 in HE of the assay, primer upstream from the PU this region was detec any amplicons detect indicating endogenou

Figure 4.7: Endogeno the PU.1 motif in vi

endogenous LIMD1 and with 20% FCS following with formaldehyde, n immunoprecipitated wit DNA purified and PCR a encompassing IR5 yield for further upstream of

LIMD1 promoter in vivo

precipitation, PU.1 specifically co-immunoprec binding motif within IR5 of the LIMD1 promot ter was used as a positive control (Brugnoli e tent with the result from the ChIP assay w

HEK 293T cells (Figure 4.6). As a further cont imers incorporating a region of the LIMD1

e PU.1 motif (US LIMD1) were also included. tected from the co-immunoprecipitated DNA, tected following precipitation with the isotype nous PU.1 specifically binds to the LIMD1 prom

genous PU.1 binds to the LIMD1 promoter th n vivo. (A) U937 human monocytic leukameic

and PU.1 as detected by Western blot. (B) U937 ce ing 24 hour serum starvation. Protein and DNA we , nuclei extracted and DNA sheared by soni with an anti-PU.1 or control IgG antibody and co- CR analysed. Primers encompassing the CD11b an

ielded amplicons, however no amplicon was produ of IR5 (US LIMD1), showing endogenous PU.1 a

vivo.

precipitated a region moter (Figure 4.7B). oli et al., 2009). This y with exogenously control for specificity promoter further ed. No amplicon for DNA, and neither were pe control antibody, romoter in vivo.

r that encompasses

meic cells express both 7 cells were stimulated were then crosslinked sonication. PU.1 was -immunoprecipitated and LIMD1 promoter roduced using primers .1 associates with the

4.3.2 PU.1 binds the IR5 region of the LIMD1 Promoter in vitro as