Jan Kadlec

The Nonspecific Lethal Complex Is a Transcriptional Regulator in Drosophila

Here, we report the biochemical characterization of the nonspecific lethal (NSL) complex (NSL1, NSL2, NSL3, MCRS2, MBD-R2, and WDS) that associates with the histone acetyltransferase MOF in both Drosophila and mammals. Chromatin immunoprecipitation-Seq analysis revealed association of NSL1 and MCRS2 with the promoter regions of more than 4000 target genes, 70% of these being actively transcribed. This binding is functional, as depletion of MCRS2, MBD-R2, and NSL3 severely affects gene expression genome wide. The NSL complex members bind to their target promoters independently of MOF. However, depletion of MCRS2 affects MOF recruitment to promoters. NSL complex stability is interdependent and relies mainly on the presence of NSL1 and MCRS2. Tethering of NSL3 to a heterologous promoter leads to robust transcription activation and is sensitive to the levels of NSL1, MCRS2, and MOF. Taken together, we conclude that the NSL complex acts as a major transcriptional regulator in Drosophila.

RNA Clamping by Vasa Assembles a piRNA Amplifier Complex on Transposon Transcripts

Germline-specific Piwi-interacting RNAs (piRNAs) protect animal genomes against transposons and are essential for fertility. piRNAs targeting active transposons are amplified by the ping-pong cycle, which couples Piwi endonucleolytic slicing of target RNAs to biogenesis of new piRNAs. Here, we describe the identification of a transient Amplifier complex that mediates biogenesis of secondary piRNAs in insect cells. Amplifier is nucleated by the DEAD box RNA helicase Vasa and contains the two Piwi proteins participating in the ping-pong loop, the Tudor protein Qin/Kumo and antisense piRNA guides. These components assemble on the surface of Vasas helicase domain, which functions as an RNA clamp to anchor Amplifier onto transposon transcripts. We show that ATP-dependent RNP remodeling by Vasa facilitates transfer of 5 sliced piRNA precursors between ping-pong partners, and loss of this activity causes sterility in Drosophila. Our results reveal the molecular basis for the small RNA amplification that confers adaptive immunity against transposons.

Unusual bipartite mode of interaction between the nonsense‐mediated decay factors, UPF1 and UPF2

Nonsense‐mediated decay (NMD) is a eukaryotic quality control mechanism that degrades mRNAs carrying premature stop codons. In mammalian cells, NMD is triggered when UPF2 bound to UPF3 on a downstream exon junction complex interacts with UPF1 bound to a stalled ribosome. We report structural studies on the interaction between the C‐terminal region of UPF2 and intact UPF1. Crystal structures, confirmed by EM and SAXS, show that the UPF1 CH‐domain is docked onto its helicase domain in a fixed configuration. The C‐terminal region of UPF2 is natively unfolded but binds through separated α‐helical and β‐hairpin elements to the UPF1 CH‐domain. The α‐helical region binds sixfold more weakly than the β‐hairpin, whereas the combined elements bind 80‐fold more tightly. Cellular assays show that NMD is severely affected by mutations disrupting the beta‐hairpin binding, but not by those only affecting alpha‐helix binding. We propose that the bipartite mode of UPF2 binding to UPF1 brings the ribosome and the EJC in close proximity by forming a tight complex after an initial weak encounter with either element.

Improving diffraction by humidity control: a novel device compatible with X-ray beamlines

Dehydration of protein crystals is rarely used, despite being a post-crystallization method that is useful for the improvement of crystal diffraction properties, as it is difficult to reproduce and monitor. A novel device for hydration control of macromolecular crystals in a standard data-collection environment has been developed. The device delivers an air stream of precise relative humidity that can be used to alter the amount of water in macromolecular crystals. The device can be rapidly installed and is fully compatible with most standard synchrotron X-ray beamlines. Samples are mounted in cryoloops and the progress of dehydration can be monitored both optically and by the acquisition of diffraction images. Once the optimal hydration level has been obtained, cryocooling is easy to achieve by hand or by using a sample changer. The device has been thoroughly tested on several ESRF beamlines and is available to users.

Structural basis for MOF and MSL3 recruitment into the dosage compensation complex by MSL1

The male-specific lethal (MSL) complex is required for dosage compensation in Drosophila melanogaster, and analogous complexes exist in mammals. We report structures of binary complexes of mammalian MSL3 and the histone acetyltransferase (HAT) MOF with consecutive segments of MSL1. MSL1 interacts with MSL3 as an extended chain forming an extensive hydrophobic interface, whereas the MSL1-MOF interface involves electrostatic interactions between the HAT domain and a long helix of MSL1. This structure provides insights into the catalytic mechanism of MOF and enables us to show analogous interactions of MOF with NSL1. In Drosophila, selective disruption of Msl1 interactions with Msl3 or Mof severely affects Msl1 targeting to the body of dosage-compensated genes and several high-affinity sites, without affecting promoter binding. We propose that Msl1 acts as a scaffold for MSL complex assembly to achieve specific targeting to the X chromosome.

Molecular basis for the regulation of the H3K4 methyltransferase activity of PRDM9.

PRDM9, a histone lysine methyltransferase, is a key determinant of the localization of meiotic recombination hot spots in humans and mice and the only vertebrate protein known to be involved in hybrid sterility. Here, we report the crystal structure of the PRDM9 methyltransferase domain in complex with a histone H3 peptide dimethylated on lysine 4 (H3K4me2) and S-adenosylhomocysteine (AdoHcy), which provides insights into the methyltransferase activity of PRDM proteins. We show that the genuine substrate of PRDM9 is histone H3 lysine 4 (H3K4) and that the enzyme possesses mono-, di-, and trimethylation activities. We also determined the crystal structure of PRDM9 in its autoinhibited state, which revealed a rearrangement of the substrate and cofactor binding sites by a concerted action of the pre-SET and post-SET domains, providing important insights into the regulatory mechanisms of histone lysine methyltransferase activity.

Structural Analysis of the Kansl1/Wdr5/Kansl2 Complex Reveals that Wdr5 is Required for Efficient Assembly and Chromatin Targeting of the Nsl Complex.

The subunits of the nonspecific lethal (NSL) complex, which include the histone acetyltransferase MOF (males absent on the first), play important roles in various cellular functions, including transcription regulation and stem cell identity maintenance and reprogramming, and are frequently misregulated in disease. Here, we provide the first biochemical and structural insights into the molecular architecture of this large multiprotein assembly. We identified several direct interactions within the complex and show that KANSL1 acts as a scaffold protein interacting with four other subunits, including WDR5, which in turn binds KANSL2. Structural analysis of the KANSL1/WDR5/KANSL2 subcomplex reveals how WDR5 is recruited into the NSL complex via conserved linear motifs of KANSL1 and KANSL2. Using structure-based KANSL1 mutants in transgenic flies, we show that the KANSL1-WDR5 interaction is required for proper assembly, efficient recruitment of the NSL complex to target promoters, and fly viability. Our data clearly show that the interactions of WDR5 with the MOF-containing NSL complex and MLL/COMPASS histone methyltransferase complexes are mutually exclusive. We propose that rather than being a shared subunit, WDR5 plays an important role in assembling distinct histone-modifying complexes with different epigenetic regulatory roles.

The multiple Tudor domain-containing protein TDRD1 is a molecular scaffold for mouse Piwi proteins and piRNA biogenesis factors.

Piwi-interacting RNAs (piRNAs) are small noncoding RNAs expressed in the germline of animals. They associate with Argonaute proteins of the Piwi subfamily, forming ribonucleoprotein complexes that are involved in maintaining genome integrity. The N-terminal region of some Piwi proteins contains symmetrically dimethylated arginines. This modification is thought to enable recruitment of Tudor domain-containing proteins (TDRDs), which might serve as platforms mediating interactions between various proteins in the piRNA pathway. We measured the binding affinity of the four individual extended Tudor domains (TDs) of murine TDRD1 protein for three different methylarginine-containing peptides from murine Piwi protein MILI. The results show a preference of TD2 and TD3 for consecutive MILI peptides, whereas TD4 and TD1 have, respectively, lower and very weak affinity for any peptide. The affinity of TD1 for methylarginine peptides can be restored by a single-point mutation back to the consensus aromatic cage sequence. These observations were confirmed by pull-down experiments with endogenous Piwi and Piwi-associated proteins. The crystal structure of TD3 bound to a methylated MILI peptide shows an unexpected orientation of the bound peptide, with additional contacts of nonmethylated residues being made outside of the aromatic cage, consistent with solution NMR titration experiments. Finally, the molecular envelope of the four tandem Tudor domains of TDRD1, derived from small angle scattering data, reveals a flexible, elongated shape for the protein. Overall, the results show that TDRD1 can accommodate different peptides from different proteins, and can therefore act as a scaffold protein for complex assembly in the piRNA pathway.

Msl1-Mediated Dimerization of the Dosage Compensation Complex Is Essential for Male X-Chromosome Regulation in Drosophila

The Male-Specific Lethal (MSL) complex regulates dosage compensation of the male X chromosome in Drosophila. Here, we report the crystal structure of its MSL1/MSL2 core, where two MSL2 subunits bind to a dimer formed by two molecules of MSL1. Analysis of structure-based mutants revealed that MSL2 can only interact with the MSL1 dimer, but MSL1 dimerization is MSL2 independent. We show that Msl1 is a substrate for Msl2 E3 ubiquitin ligase activity. ChIP experiments revealed that Msl1 dimerization is essential for targeting and spreading of the MSL complex on X-linked genes; however, Msl1 binding to promoters of male and female cells is independent of the dimer status and other MSL proteins. Finally, we show that loss of Msl1 dimerization leads to male-specific lethality. We propose that Msl1-mediated dimerization of the entire MSL complex is required for Msl2 binding, X chromosome recognition, and spreading along the X chromosome.

Comparative Structural and Functional Analysis of Orthomyxovirus Polymerase CAP-Snatching Domains.

Orthomyxovirus Influenza A virus (IAV) heterotrimeric polymerase performs transcription of viral mRNAs by cap-snatching, which involves generation of capped primers by host pre-mRNA binding via the PB2 subunit cap-binding site and cleavage 10–13 nucleotides from the 5′ cap by the PA subunit endonuclease. Thogotoviruses, tick-borne orthomyxoviruses that includes Thogoto (THOV), Dhori (DHOV) and Jos (JOSV) viruses, are thought to perform cap-snatching by cleaving directly after the cap and thus have no heterogeneous, host-derived sequences at the 5′ extremity of their mRNAs. Based on recent work identifying the cap-binding and endonuclease domains in IAV polymerase, we determined the crystal structures of two THOV PB2 domains, the putative cap-binding and the so-called ‘627-domain’, and the structures of the putative endonuclease domains (PA-Nter) of THOV and DHOV. Despite low sequence similarity, corresponding domains have the same fold confirming the overall architectural similarity of orthomyxovirus polymerases. However the putative Thogotovirus cap-snatching domains in PA and PB2 have non-conservative substitutions of key active site residues. Biochemical analysis confirms that, unlike the IAV domains, the THOV and DHOV PA-Nter domains do not bind divalent cations and have no endonuclease activity and the THOV central PB2 domain does not bind cap analogues. On the other hand, sequence analysis suggests that other, non-influenza, orthomyxoviruses, such as salmon anemia virus (isavirus) and Quaranfil virus likely conserve active cap-snatching domains correlating with the reported occurrence of heterogeneous, host-derived sequences at the 5′ end of the mRNAs of these viruses. These results highlight the unusual nature of transcription initiation by Thogotoviruses.