Structural basis for interaction between CLAMP and MSL2 proteins involved in the specific recruitment of the dosage compensation complex in Drosophila

Abstract

Transcriptional regulators select their targets from a large pool of similar genomic sites. The binding of the Drosophila dosage compensation complex (DCC) exclusively to the male X chromosome provides insight into binding site selectivity rules. Previous studies showed that the male-specific organizer of the complex, MSL2, and ubiquitous DNA-binding protein CLAMP directly interact and play an important role in the specificity of X chromosome binding. Here we studied the highly specific interaction between the intrinsically disordered region of MSL2 and the N-terminal zinc-finger C2H2-type (C2H2) domain of CLAMP. We obtained the NMR structure of the CLAMP N-terminal C2H2 zinc finger, which has a classic C2H2 zinc-finger fold with a rather unusual distribution of residues typically used in DNA recognition. Substitutions of residues in this C2H2 domain had the same effect on the viability of males and females, suggesting that it plays a general role in CLAMP activity. The N-terminal C2H2 domain of CLAMP is highly conserved in insects. However, the MSL2 region involved in the interaction is conserved only within the Drosophila genus, suggesting that this interaction emerged during the evolution of a mechanism for the specific recruitment of the DCC on the male X chromosome in Drosophilidae.

Dimerization Activity of a Disordered N-Terminal Domain from Drosophila CLAMP Protein

“…We recently found that the region of 1–153 amino acid residues can homodimerize in the Y2H assay [ 46 ]. For detailed mapping of the CLAMP domain involved in homodimerization, we tested different variants of the N-terminal region for interaction in the Y2H assay.…”

“…To assess the folding state of the CLAMP N-terminal domain, we used NMR spectroscopy. We measured the 15 N-HSQC NMR spectrum of 15 N-labeled CLAMP 1–113 and compared it with a spectrum of CLAMP 87–153 including the zinc-finger region for which the resonance assignments were obtained previously ([ 46 ], BioMagResBank ID: 34600 Available online: (accessed on 26 December 2021). All H N chemical shifts of the 1–86 region are plotted into the interval from 7.6 to 8.6 ppm.…”

“…Tikhonova et al [ 46 ] demonstrated that the N-terminal dimerization domain facilitates a relatively weak interaction between the C2H2 domain of CLAMP and MSL2. Because CLAMP exhibits architectural properties [ 38 ], the dimerization domain might be involved in the organization of distance interactions between regulatory elements in a manner similar to the CTCF protein.…”

“… ( a ) 15 N- 1 H-HSQC spectra of D. melanogaster CLAMP 1–113 (red) and CLAMP 87–153 (blue). The amino acid assignment (performed for residues 87–153 in [ 46 ], BioMagResBank ID: 34600) is shown. ( b ) R 2 NMR relaxation times for CLAMP residues in 41–86, 89–119, and 122–151 regions.…”

MSL2 targets histone genes inDrosophila virilis

“…198 199 MSL2 is the structural component of MSLc and is usually found complexed with other members (Hallacli et al 2012). However, MSL2 has some affinity for DNA sequence in both D. melanogaster and D. virilis (Villa et al 2016(Villa et al , 2021 and CLAMP and MSL2 directly interact with each other through well-conserved domains (Tikhonova et al 2022b), suggesting that CLAMP might recruit MSL2 outside of the complex. We therefore stained chromosome spreads for another MSLc member, MSL3.…”