Structure and Function of the Su(H)-Hairless Repressor Complex, the Major Antagonist of Notch Signaling in Drosophila melanogaster

Abstract

Notch is a conserved signaling pathway that specifies cell fates in metazoans. Receptor-ligand interactions induce changes in gene expression, which is regulated by the transcription factor CBF1/Su(H)/Lag-1 (CSL). CSL interacts with coregulators to repress and activate transcription from Notch target genes. While the molecular details of the activator complex are relatively well understood, the structure-function of CSL-mediated repressor complexes is poorly defined. In Drosophila, the antagonist Hairless directly binds Su(H) (the fly CSL ortholog) to repress transcription from Notch targets. Here, we determine the X-ray structure of the Su(H)-Hairless complex bound to DNA. Hairless binding produces a large conformational change in Su(H) by interacting with residues in the hydrophobic core of Su(H), illustrating the structural plasticity of CSL molecules to interact with different binding partners. Based on the structure, we designed mutants in Hairless and Su(H) that affect binding, but do not affect formation of the activator complex. These mutants were validated in vitro by isothermal titration calorimetry and yeast two- and three-hybrid assays. Moreover, these mutants allowed us to solely characterize the repressor function of Su(H) in vivo.

Hairless-binding deficient Suppressor of Hairless alleles reveal Su(H) protein levels are dependent on complex formation with Hairless

“…In vivo , we noted only little differences amongst the three mutant alleles. With regards to fly viability and SOP formation, however, Su(H) LL appeared a somewhat weaker allele than the other two, in agreement with the residual H-binding activity this mutant displayed in yeast two-hybrid and overexpression assays [11]. All three alleles are larval to early pupal lethal, clearly demonstrating the pivotal role of Su(H) as a repressor during fly development.…”

“…This strategy was chosen to avoid possible splice defects. The three alanine substitution mutations were originally introduced in Su(H) cDNA [11]. They were shuttled into the genomic Su(H) DNA whenever possible, resulting in the loss of some of the three introns: Su(H) LL and Su(H) LLF lack intron 3, and Su(H) LLL lacks introns 2 and 3.…”

“…Recently, the crystal structure of the core Notch repressor complex, containing Su(H) and H bound to DNA, was determined [11]. Strikingly, H binds deeply into the hydrophobic core of the CTD of Su(H), thereby causing a major conformational change in Su(H) that is incompatible with NICD binding.…”

Integration of Drosophila and Human Genetics to Understand Notch Signaling Related Diseases

“…In the absence of NICD, Su(H) is bound to the co-repressor Hairless (H)[176,15,177] which in turn recruits additional co-repressors such as Groucho [TLE (Transducin-like enhancer protein) 1–6 in human, TLE5 is also referred to as AES (Amino-terminal Enhancer Of Split)] and CtBP (C-terminal Binding Protein, CTBP1–2 in human) to silence target genes [178–182]. Once NICD enters the nucleus and binds to Su(H), H is no longer able to bind to Su(H)[183]. The active NICD-Su(H)-Mam complex further recruits transcriptional co-activators such as the histone acetyltransferase CBP [CREB (cAMP response element binding protein)-binding protein]/p300 [nejire (nej) in Drosophila, EP300 (E1A binding protein P300) and CREBBP (CREB-binding protein) in human] to initiate transcription of downstream target genes [71,184].…”

“…Instead, two structurally unrelated co-repressors, KyoT2 (encoded by the FHL1 gene in human)[185] and SHARP [SMRT/HDAC1 Associated Repressor Protein, encoded by the SPEN (SPlit ENds family transcriptional repressor) gene in human, also called Mint] [186,187] play the same function, binding to RBPJ and further recruiting additional corepressors to silence transcription[188]. Interestingly, Hairless and KyoT2/SHARP bind to RBPJ through different molecular mechanisms[183,189,190], suggesting that these genes were integrated into the Notch pathway independently through convergent evolution.…”