Chromatin and origins Supp info.
Aggarwal and Calvi
Supplementary Methods
Plasmid construction: Tether Target 1 or P{w+mC, TT1}, hereafter referred to as
TT1, was made by inserting the 3.8 kb Sal I fragment from the 3rd chorion locus into the
pUAST P-element vector 1. For construction of the heat- inducible Gal4 fusion vector
P{w+mC, hsp70:Gal4DBD}, the first 147 amino acids of Gal4, which contains the DNA
binding domain (DBD), was PCR amplified from pSG424 2, and inserted into the EcoR I /
Not I downstream of hsp70 promoter in the P-element pCaSpeR-Hs vector 3. Rpd3
(CG7471) was PCR amplified from a D. melanogaster embryonic cDNA library 4. The
sequence of this cDNA matches genbank sequence Q94517 5. This cDNA was inserted inframe downstream of Gal4(DBD) in Kpn I / Xba I site to make P{w+mC,
hsp70:Gal4DBDRpd3}. To make P{w+mC, hsp70:Gal4DBD:HAT1}, the coding region of
HAT1 (the chameau gene, CG5229) was PCR amplified and subcloned into the Bgl II/Not
I sites of P{w+mC, hsp70:Gal4DBD}. P elements were transformed into y w67c23 by standard
methods 6. Fly Strain P{w+mC , hsp70-Gal4(DBD):Pc} was a kind gift from Dr. Dirke
Beuchle, Max-Planck-Institute 7.
Tethering measurements: For calculation of the ratios of TT1 to endogenous 3rd locus,
the total number of nuclei examined for induced and uninduced respectively were:
GAL4DBD 180 and 153; GAL4DBD:Rpd3 278 and 250; GAL4DBD:Rpd3H137A 96 and 90;
GAL4DBD:HAT1 424 and 409; GAL4DBD:Pc 365 and 385. For the frequency of TT1
detection, the number of nuclei examined for induced and uninduced was: GAL4DBD 280
and 367; GAL4DBD:Rpd3 394 and 382; GAL4DBD:Rpd3H137A 110 and 83; GAL4DBD:HAT1
582 and 486; GAL4DBD:Pc 347 and 562. An unpaired students t test was used to evaluate
the significance of the difference between control and experimental samples. A test of our
GAL4DBD:Rpd3 and GAL4DBD:RPD3H137A in a mammalian cell luciferase assay showed
1
2/17/16
Chromatin and origins Supp info.
Aggarwal and Calvi
that both could repress transcription from the thymidine kinase promoter consistent with
previous reports 8,9.
Rpd3 mitotic clones: Mitotic clones were generated by standard methods 10. We observed
two types of GFP negative clones in amplification stage egg chambers. Large clones (10100’s cells/clone), which represent recombination events that occurred very early in
oogenesis, had nuclei with aberrant morphology likely due to severe deleterious and
pleiotropic effects of Rpd3m5-5 (data not shown). In contrast, clones with fewer cells (1-10
cells), which represent recombination events later in oogenesis, had nuclei that appeared
normal, or sometimes larger, than neighboring wild-type cells. We focused on these small
clones generated late in oogenesis as most informative for proximal effects on replication.
Quantification of fluorescent antibody labeling: Fluorescent intensity was quantified by
capturing non-saturated images with a Leica SP confocal followed by analysis with the
Leica TCS-NT quantification software. For Fig. S2, images were captured using a 100x
objective with a 2x zoom resulting in a voxel size of 50 nm. At least 15 nuclei were
analyzed for each measure.
ChIP PCR: The following primer sets spanning the 50 kb region to either side of ACE3
were used and designated as –50 kb, -10kb, ACE3, Ori-, +10 kb, +50 kb. Primers to nondevelopmentally amplified sequence from 6C on the X chromosome were used as a control.
The sequences of the primers used are:
–50 kb primers: CCATTCGCGACTGTGGGCGACTGGCC and
CTGGCCAACTCATTGAACCGGTGCCGTTCG
–10kb primers: CGAAGATGGGCTGGTCCTCTTGTCTCGC and
GCCGAGTCAACGAGGTGAGCACATCG;
2
2/17/16
Chromatin and origins Supp info.
Aggarwal and Calvi
ACE3 primers: GGTACCCTGAGCCTGGCCAACATC and
CCGCATAGTTTCGATCAGTATTGC;
Ori- primers: AAAGCTAAAACTAAATTAATTTGTGGGG and
GGTTCCAGCCGGTTTTTCTGATAAAACC;
+10kb primers: CAGTGTCGACAATTGGCGCTTCAC and
CCAGAAACGCTCGCCGTTCAGTGTAC;
+50kb primers: CGGAGTGGGCGTGGCGTGCCAACAATTTGC and
GCATTCAGCAGCCGCAGGATTGTGCCCAGC;
6C primers: ATGGGCAGGAATCGCAACCTGCA and
TGACAGCTGCTTGTGCGGAACTT.
ChIP PCR reactions (50 l) contained input or pellet ChIP DNA diluted in 5 fold
M of
ThermoPol
each primer, andVent (exo-) DNA polymerase (New England BioLabs). PCR was in the
linear range of amplification, which was empirically determined. Different dilutions of the
PCR products were analyzed on ethidium bromide stained agarose gels and quantified
against a standard curve using a MultiImager (Alpha Innotech Corporation) and
ChemiImager V5.5 (Alpha Innotech Corporation) softwares. Fold enrichment in the
pellet versus the input was normalized to pellet/input ratios for the 6C control locus. This
controls for both primer efficiency and differential copy number in the input. Figure 2
represents the averages and standard deviations for two independent immunoprecipitations
and four PCR reactions for Ori- and ACE3, and three to four PCR reactions for other 3rd
chromosome primers.
3
2/17/16
Chromatin and origins Supp info.
Aggarwal and Calvi
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Brand, A. & Perrimon, N. Targeted gene expression as a means of altering cell fates
and generating dominant phenotypes. Development 118, 401-415 (1993).
Sadowski, I. & Ptashne, M. A vector for expressing GAL4(1-147) fusions in
mammalian cells. Nucleic Acids Res 17, 7539 (1989).
Thummel, C. S., Boulet, A. M. & Lipshitz, H. D. Vectors for Drosophila P-elementmediated transformation and tissue culture transfection. Gene 74, 445-456. (1988).
Brown, N. H. & Kafatos, F. C. Functional cDNA libraries from Drosophila
embryos. J Mol Biol 203, 425-37 (1988).
De Rubertis, F. et al. The histone deacetylase RPD3 counteracts genomic silencing
in Drosophila and yeast. Nature 384, 589-91 (1996).
Spradling, A. C. & Rubin, G. M. Transposition of cloned P elements into
Drosophila germ line chromosomes. Science 218, 341-347 (1982).
Beuchle, D., Struhl, G. & Muller, J. Polycomb group proteins and heritable
silencing of Drosophila Hox genes. Development 128, 993-1004 (2001).
Hassig, C. A. et al. A role for histone deacetylase activity in HDAC1-mediated
transcriptional repression. Proc Natl Acad Sci U S A 95, 3519-24 (1998).
Kadosh, D. & Struhl, K. Histone deacetylase activity of Rpd3 is important for
transcriptional repression in vivo. Genes Dev 12, 797-805 (1998).
Xu, T. & Rubin, G. M. Analysis of genetic mosaics in developing and adult
Drosophila tissues. Development 117, 1223-37 (1993).
4
2/17/16