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. 2013 Jul 15;27(14):1551-6.
doi: 10.1101/gad.214585.113.

The CLAMP protein links the MSL complex to the X chromosome during Drosophila dosage compensation

Marcela M L Soruco  1 , Jessica Chery, Eric P Bishop, Trevor Siggers, Michael Y Tolstorukov, Alexander R Leydon, Arthur U Sugden, Karen Goebel, Jessica Feng, Peng Xia, Anastasia Vedenko, Martha L Bulyk, Peter J Park, Erica Larschan
Affiliations

The CLAMP protein links the MSL complex to the X chromosome during Drosophila dosage compensation

Marcela M L Soruco et al. Genes Dev. .

Abstract

The Drosophila male-specific lethal (MSL) dosage compensation complex increases transcript levels on the single male X chromosome to equal the transcript levels in XX females. However, it is not known how the MSL complex is linked to its DNA recognition elements, the critical first step in dosage compensation. Here, we demonstrate that a previously uncharacterized zinc finger protein, CLAMP (chromatin-linked adaptor for MSL proteins), functions as the first link between the MSL complex and the X chromosome. CLAMP directly binds to the MSL complex DNA recognition elements and is required for the recruitment of the MSL complex. The discovery of CLAMP identifies a key factor required for the chromosome-specific targeting of dosage compensation, providing new insights into how subnuclear domains of coordinate gene regulation are formed within metazoan genomes.

Keywords: Drosophila; chromatin; dosage compensation; zinc finger protein.

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Figures

Figure 1.
Figure 1.
CLAMP localizes precisely at MREs at high levels when colocalized with the MSL complex. (A) Occupancy relationships calculated from input-normalized CLAMP ChIP-seq from male SL2 cells (Larschan et al. 2012) and previously defined CESs (Alekseyenko et al. 2008) (two biological replicates). MSL complex-binding sites ranked based on MSL complex occupancy from ChIP-seq read counts (X-axis; strongest on the left and weakest on the right) (Alekseyenko et al. 2008). The Y-axis indicates the position relative to the center of the MSL complex sites. Each red dot represents the position of a CLAMP-binding site that is within 1 kb of an MSL complex-binding site. The intensity of the red color is proportional to CLAMP occupancy (see the Materials and Methods). The intensity of the blue color is proportional to similarity of the individual MRE sequence to the consensus sequence. (B) Examples of CLAMP and MSL complex occupancy at three CESs are shown from SL2 cells. Input-normalized read counts are shown on a log scale for CLAMP and the MSL complex.
Figure 2.
Figure 2.
CLAMP directly binds to MRE sequences. (A, top) PWM for the original MRE motif (Alekseyenko et al. 2008). (Bottom) PWMs were derived from CLAMP ChIP-seq data from SL2 cells. MEME was used to analyze 200-bp regions flanking 500 CLAMP occupancy sites that were located at the 3′ ends of active genes on the X chromosome. (B) PBM analysis resulted in direct in vitro binding specificities of the C-terminal CLAMP four (top) and six (bottom) zinc finger fragments.
Figure 3.
Figure 3.
CESs have three classes of CLAMP occupancy patterns. (A) CLAMP or MSL2 occupancy patterns (reads per kilobase per million mapped reads [RPKM] normalized to input) are shown within a 2-kb window centered at the MRE for each member of each subclass of CESs (265 total CESs). (1) Group A sites are highly MSL-dependent. (2) Group B sites are partially MSL-dependent. (3) Group C sites are independent of the MSL complex. (B) Average profiles of CLAMP occupancy (RPKM) normalized to input are centered at group A, group B, or group C CES peaks and are shown compared with average occupancy at all CLAMP sites on the X or autosomes (log2 scale).
Figure 4.
Figure 4.
Three subclasses of CESs differ in their number of tandem MREs and two-dimensional clustering along the X chromosome. (A) Analysis of the number of tandem MREs within each subclass of CESs. (B) The locations of CESs from each subclass of sites are plotted to allow their relative positions along the length of the X chromosome to be determined. The Y-axis is the position along the length of the X chromosome, and the X-axis shows the percentage of sites plotted. The positions of the roX loci are shown as green lines. (C) Model for CLAMP and roX RNAs functioning together during X identification: (1) CLAMP binds to MREs within group B and group C independent of the MSL complex, and roX RNAs are transcribed independent of MSL complex recruitment. (2) The MSL complex is likely to be cotranscriptionally assembled at roX loci. CLAMP tethers the MSL complex to CESs, including roX loci. The MSL complex catalyzes H4K16ac, thereby opening chromatin and increasing the accessibility of MRE sequences for CLAMP recognition. Next, increased levels of CLAMP associate with CESs and thereby recruit additional MSL complexes via synergistic interactions. (3) The MSL complex spreads to additional CESs, potentially stabilized by CLAMP, thereby initiating additional synergistic interactions.
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