1. Volume 11, Issue 4, Pages 977-986 (April 2003)
Sequence-Specific Targeting of Drosophila roX Genes by the MSL Dosage Compensation Complex 
Yongkyu Park, Gabrielle Mengus, Xiaoying Bai, Yuji Kageyama, Victoria H Meller, Peter B Becker, Mitzi I Kuroda 
Molecular Cell 
Volume 11, Issue 4, Pages (April 2003)
DOI: /S (03)

2. Figure 1
The DNase I Hypersensitive Region of roX2 Is Located Downstream of the Major 3′ Processing Site of Most roX2 Transcripts
(A) The gene structure of roX2 includes Promoter (P), Exon 1 (110 bp), Intron (545 bp), Exon 2 (430 bp), and DHS (270 bp) regions. The major and minor 3′ ends are indicated as three arrowheads within the line representing the gene. Thick line, major transcript; dotted line, 3′ region included in minor transcripts. Hatch box, 30 nt similarity of unknown function between roX1 and roX2 (Franke and Baker, 1999); black box, 110 bp segment containing islands of conserved sequences (Figure 5A).
(B) RT-PCR to map the 5′ end of roX2. RT-PCR products using primers indicated in Figure 1A show the major spliced form. 1, 100 bp ladder; 2–11, RT-PCR; 12, PCR using roX2 genomic fragment as template.
(C) Northern analysis of roX2 RNA from adult flies. The membranes were exposed for 36 hr (for roX2 probes of different sizes but approximately the same specific activity) or 24 hr (for rp49 as loading control). The weaker signal from the exon1 probe could be due to its smaller size. Left numbers, RNA ladder; M, male; F, female.
(D) DNase I hypersensitivity within the roX2 locus. DNase I cleavages were mapped within a BglII fragment containing the roX2 gene, schematized to the left (open box, +1 indicates the approximate 5′ end of the gene). Nuclei from female (left) or male (right) adult flies were treated with increasing concentrations of DNase I. DNA was isolated and digested to completion with BglII. DNase I cleavages were revealed by Southern blot using a probe adjacent to a BglII site located 2466 bp downstream of the roX2 transcription start site. The double-headed arrow highlights male-specific DNase I hypersensitivity in roX2 chromatin. The numbers to the right correspond to a DNA size marker and indicated sizes in bp.
Molecular Cell  , DOI: ( /S (03) )

3. Figure 2 The DHS Region of roX2 Is an MSL Binding Site In Vivo
Polytene chromosome squashes from transgenic larvae immunostained with rabbit anti-MSL1 (red) and counterstained with DAPI (blue).
(A) Male nucleus containing a monomer of the DHS fragment [roX2DHS-59D].
(B) Male nucleus containing a multimer [roX2DHS-91A].
(C) Monomer [roX2DHS-47B] in a female nucleus carrying [Hsp83MSL2] and mutant for msl3.
(D) Male nucleus containing a 110 bp roX2 monomer [roX2CR-78E].
Molecular Cell  , DOI: ( /S (03) )

4. Figure 3
roX RNA Is Assembled and Functional without a Linked MSL Binding Site
All experiments were performed in a roX1 roX2 genetic background. Red, rabbit anti-MSL1; blue, DAPI.
(A) Rescue frequency (male:female ratio) of roX1 roX2 mutants by four different [H83roX2ΔDHS] transgenic lines.
(B–D) MSL complexes containing roX2ΔDHS RNA paint the X chromosome although the [H83roX2ΔDHS] insertion sites show no or weak binding of MSL proteins because of the absence of the DHS region.
(E–H) Competition for cis-spreading from [GMroX2-97F] (arrow) with roX cDNA constructs (arrowheads). (E) [roX1DHS-31F]; (F) [roX2DHS-59D]; (G) [H83roX1ΔDHS-92C]; (H) [H83roX2ΔDHS-83C]. [roXDHS] transgenes alone cannot compete for cis-spreading from [GMroX2-97F]. However, [H83roXΔDHS] transgenes show strong competition.
Molecular Cell  , DOI: ( /S (03) )

5. Figure 4 roX2 Genes from Other Drosophila Species
(A) The divergence distance of the three species. Mya, million years.
(B) Low-stringency Northern analysis using roX2 and rp49 probes from D. melanogaster.
(C) Sequence similarity of roX2 from D. melanogaster, D. simulans, and D. erecta analyzed using the GCG program.
Molecular Cell  , DOI: ( /S (03) )

6. Figure 5
Consensus Sequences within the DHS Are Important for Binding of MSL Complex
(A) Consensus sequences (gray box) common to the MSL binding sites of roX1 and roX2. Conserved (1, 2, 4, and 5) and nonconserved (3) sequences were mutagenized by five base substitutions. The degree of MSL binding was determined by comparing binding to wild-type (wt) and mutant transgenes of [roX2DHS] in the same nucleus. The scores represent comparisons of multiple combinations of wt and mutant transgenes analyzed independently by four different people.
(B) Immunostaining with anti-MSL1 antibodies (red) reveals binding to a wild-type [roX2DHS] (W, arrow) at 21E (panels 1–5), or to mutant [roX2DHS] (M, arrowhead) at 60E (mutant 1), 29B (mutant 2), 30C (mutant 3), 92F (mutant 4), or 100D (mutant 5).
(C) Two blocks of consensus sequences are not enough to attract MSL complex. Blue blocks (1 and 5) are regions that show a mild effect when mutagenized, and red blocks (2 and 4) are regions that show a strong effect. 1B7, 11D1, and 12E8 were candidate chromatin entry sites that were unable to attract MSL proteins to autosomal transgenes.
Molecular Cell  , DOI: ( /S (03) )

7. Figure 6 MSL Binding on roX DHS Sequences Is Dependent on roX RNA
Red, rabbit anti-MSL1; blue, DAPI staining. (A) roX1 roX2 double mutant male nucleus showing weak MSL binding to the X chromosome and ectopic binding to autosomal sites and heterochromatin (arrowhead). (A–F) [roX2DHS-59D] transgenic males. In (B), the transgene (arrow) is not recognized by MSL proteins in the roX1 roX2 double mutant male. (A) and (B) show the same nucleus, with (A) at lower magnification. (C and D) [roX2DHS-59D] is recognized by MSL complexes in roX1 or roX2 single mutant males. (E and F) Recognition of [roX2DHS-59D] is restored in a roX1− roX2− male by expression of [H83roX2ΔDHS-83C] or [H83roX1ΔDHS-23B].
(G–K) [roX1DHS-31F] transgenic males. In (G), the transgene (arrow) is not recognized by MSL proteins in the roX1 roX2 double mutant male. Stars indicate sites of ectopic autosomal binding of MSL complexes that occurs frequently in double mutant males. (H and I) [roX1DHS-31F] is recognized by MSL complexes in roX1 or roX2 single mutant males. (J and K) Recognition of [roX1DHS-31F] is restored in a roX1− roX2− male by expression of [H83roX2ΔDHS-83C] or [H83roX1ΔDHS-23B].
Molecular Cell  , DOI: ( /S (03) )