van Steensel et al, Supplementary Information
van Steensel et al.
Supplementary information
METHODS
Plasmids. Plasmid sequences may be obtained from
http://blocks.fhcrc.org/DamID. Vectors for expression of myc-tagged Dam and Dam–HP1
were described previously [B. van Steensel and S. Henikoff (2000), Nature Biotechnol.
18:424-428]. A cDNA encoding full-length Sir2 (GenBank accession AF068758) was
obtained by PCR amplification from a Drosophila ovary cDNA library (kindly provided
by S. Parkhurst) and cloned into pCMycDam, resulting in plasmid pSir2–MDam.
Sequencing of the cloned PCR product revealed that six nucleotides encoding Phe289 and
Gln290 are missing. This polymorphism is also present in a genomic sequence (GenBank
AE003639). Dam was fused to the C terminus of Sir2, because addition of green
fluorescent protein to this end of yeast Sir2 did not interfere with correct subnuclear
targeting of Sir2 [G. Cuperus et al(2000), EMBO J. 19:2641-2651]). Full-length GAF
(the 519 amino acid isoform [Benyajati, 1997, critical]) was amplified from plasmid
pAR-GAGA [W.C. Soeller et al(1993), Mol. Cell. Biol. 13:7961-7970] (kindly provided
by T. Tsukiyama) and cloned either into pCMycDam (resulting in pGAF-MDam) or
pNDamMyc (resulting in pDamM-GAF).
Microarray construction. Because Drosophila genomic DNA microarrays were
not available, we used arrays assembled by the Northwest Flychip Consortium.
Microarray construction and hybridization protocols were modified and optimized from
those described elsewhere [J.L. DeRisi et al(1997), Science 278:680-686]. Drosophila
microarrays were constructed employing a set of 192 cDNAs randomly selected from the
LD and CK EST libraries (Berkeley Drosophila Genome Project and Research Genetics,
Huntsville, AL) [G.M. Rubin et al(2000), Science 287:2222-2224; C.C. Kopczynski et
al(1998), Proc. Natl. Acad. Sci. USA 95:9973-9978], together with 140 cDNA and 20
genomic DNA fragments provided by members of the Northwest Fly Consortium. Each
clone insert was individually PCR-amplified and each product was verified as unique via
gel electrophoresis. PCR products were purified using ArrayIt 96-well PCR purification
1
van Steensel et al, Supplementary Information
kits (TeleChem International, Sunnyvale, CA) and mechanically "spotted" in 3SSC (450
mM sodium chloride and 45 mM sodium citrate, pH 7.0) onto poly-lysine coated
microscope slides using an OmniGrid high-precision robotic gridder (GeneMachines, San
Carlo, CA).
Normalization procedure for microarray data. There are two subsequent
normalization procedures in our protocol. The first and most important normalization is
the normalization/correction for local chromatin accessibility [see also van Steensel and
Henikoff (2000), Nature Biotechnol. 18:424-428]. This is done during the actual
experiment by using methylated DNA from Dam-only transfected cells as a reference
probe. This reference probe is mixed and co-hybridized with the experimental probe from
cells expressing the Dam-fusion protein (a typical approach for microarray experiments).
We like to emphasize that both probes are prepared from genomic DNA from cells with
the same genomic makeup (except for the transfected plasmids). Hence, if after
purification of methylated fragments a particular sequence is more abundant in the
experimental probe than in the reference probe, then this must mean that this sequence
was more heavily methylated by the Dam-fusion protein than by Dam alone, and thus that
this sequence is a target of the chromatin protein that was fused to Dam. This is true for
single-copy loci as well as for repetitive sequences in the genome.
The second normalization is a numerical adjustment to allow comparison between
independent experiments. The measured Cy3:Cy5 ratios (raw data) have an arbitrary
scale, because they depend on many factors, including the probe labeling efficiencies and
the gain settings of the scanner. Using an internal standard on each microarray allows
correction for these factors. We chose to use 16 spots of total genomic DNA on each
array as the internal standard, but we could have chosen any other standard (such as the
average value of all cDNAs on the array). Importantly, this choice does not affect the
final outcome of the experiments, since the Cy3:Cy5 ratios of all loci are multiplied by
the same constant. We preferred genomic DNA spots on the microarray as the
normalization standard, because the final normalized Cy3:Cy5 values are intuitively
meaningful: a value >1 means that the binding to a locus is more than the average binding
level in the entire genome; a value <1 means that the binding was less than the average
2
van Steensel et al, Supplementary Information
binding level in the entire genome.
Immunocytochemistry. Kc cells were transfected with plasmids encoding Damfusion protein under a heat-shock inducible promoter and grown on microscope
coverslips. After 18 h, cells were heat-shocked at 37 °C for 2 h, followed by 5 h recovery
at 25 °C. Immunocytochemistry was performed as described [S. Henikoff et al(2000),
Proc. Natl. Acad. Sci. USA 97:716-721], using a rabbit polyclonal antibody against the
hinge domain of HP1 [J. Smothers and S. Henikoff, in press]. Dam fusion proteins were
detected using mouse monoclonal antibody 9E10 (Santa Cruz Biotechnology, Santa Cruz,
CA) against the myc epitope, which is present as a linker peptide between Dam and its
fusion partners.
3