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.

Physical interaction between MSL2 and CLAMP assures direct co-operativity and prevents competition at composite binding sites

“…Weaker binding sites contain degenerate MRE motifs dominated by GA repeats. These data support earlier conclusions from low-resolution studies on polytene chromosomes about the existence of a hierarchy of binding sites, where sites of higher affinity are primary attractants for MSL proteins and sites of lower affinity profit from local TF enrichment (24,30).…”

“…The new profiles reveal more than twice as many MSL2 binding sites than corresponding Chromatin Immunoprecipitation (ChIP) profiles, which are, remarkably, all on the X chromosome. The improved profiling method not only visualizes HAS, but also degenerate sites of lower affinity that are still restricted to the X (24). Interestingly, we observed only a minimal overlap between the in vitro and in vivo binding sites.…”

“…C&R yielded twice as many MSL2 binding sites (709) than corresponding ChIP profiles. Apparently, the improved profiling method not only visualizes HAS, but also degenerate sites of lower affinity that are nevertheless restricted to the X (24,30). As such sites have not been previously characterized, we divided the total set of peaks into thirds according to their binding strength and performed motif searches using MEME.…”

Interaction of MLE with CLAMP zinc finger is involved in proper MSL proteins binding to chromosomes in Drosophila

“…MSL2-CLAMP interaction [20,21,22] MSL2 DNA-binding [13,14] -interacted regions -role of interaction in MSL complex localization MRE [11,12] CLAMP DNA-binding [17,18] HAS domains [40], a helicase associated 2 (HA2) domain, and an oligonucleotide-binding (OB)-fold domain. The region between 105 and 1158 aa forms the core of the MLE protein (figure 2a).…”

“…The CXC domain binds with high specificity to MREs in vitro [14][15][16]. Ubiquitous transcription factor chromatin-linked adaptor for MSL proteins (CLAMP) binds also to GArich sequences (MREs) in most of the HAS/CES [17][18][19] and directly interacts with MSL2's CLAMP binding domain (CBD), located distally of its CXC domain [20][21][22]. It is proposed that the interaction between CLAMP and MSL2 is important for the specific binding of the MSL complex to the male X chromosome [20,21,23].…”