Structural mechanism of extranucleosomal DNA readout by the INO80 complex

The nucleosomal landscape of chromatin depends on the concerted action of chromatin remodelers. The INO80 remodeler specifically places nucleosomes at the boundary of gene regulatory elements, which is proposed to be the result of an ATP-dependent nucleosome sliding activity that is regulated by extranucleosomal DNA features. Here, we use cryo–electron microscopy and functional assays to reveal how INO80 binds and is regulated by extranucleosomal DNA. Structures of the regulatory A-module bound to DNA clarify the mechanism of linker DNA binding. The A-module is connected to the motor unit via an HSA/post-HSA lever element to chemomechanically couple the motor and linker DNA sensing. Two notable sites of curved DNA recognition by coordinated action of the four actin/actin-related proteins and the motor suggest how sliding by INO80 can be regulated by extranucleosomal DNA features. Last, the structures clarify the recruitment of YY1/Ies4 subunits and reveal deep architectural similarities between the regulatory modules of INO80 and SWI/SNF complexes

Examining the generalizability of research findings from archival data

This initiative examined systematically the extent to which a large set of archival research findings generalizes across contexts. We repeated the key analyses for 29 original strategic management effects in the same context (direct reproduction) as well as in 52 novel time periods and geographies; 45% of the reproductions returned results matching the original reports together with 55% of tests in different spans of years and 40% of tests in novel geographies. Some original findings were associated with multiple new tests. Reproducibility was the best predictor of generalizability—for the findings that proved directly reproducible, 84% emerged in other available time periods and 57% emerged in other geographies. Overall, only limited empirical evidence emerged for context sensitivity. In a forecasting survey, independent scientists were able to anticipate which effects would find support in tests in new samples

Structural elucidation of modular regulation of the Swi2/Snf2 ATPases Mot1 and Ino80

In eukaryotes, chromatin – the DNA packaged by nucleosomes and other bound proteins - is constantly reshaped by energy-dependent processes that facilitate accessibility of DNA for the replication, repair and transcription machinery. Swi2/Snf2 helicases translate energy derived from ATP-hydrolysis into DNA minor groove translocation resulting in either tracing DNA or pumping or pulling it to disrupt protein:DNA interactions, termed “chromatin remodeling”. The transcription regulator Mot1 is a single-subunit Swi2/Snf2 ATPase that removes TBP from the TATA box at the DNA promoter, thus recycling it and enabling a redistribution to other promoters. Despite a wealth of biochemistry, the chemo-mechanical details of the TBP removal were unknown. In the first publication, we present the crystal structure of near full-length Mot1 in an autoinhibited resting state. This allowed insight into the interaction between N-terminal HEATrepeat arch and C-terminal ATPase in nucleotide-free state. In the second publication, we employed cryogenic electron microscopy (cryo-EM) to determine five structures of Mot1 bound to its TBP:DNA substrate with different ATP analogues. We could therefore dissect the stepwise dissociation of TBP from DNA in molecular detail and analyze the structure and function of the outermost C-terminal “bridge” element as an allosteric regulator of the remodeling activity of Mot1. Ultimately, we arrived at a model that involves a short-range, non-processive DNA translocation by Mot1, including bending and rotation of the DNA. This is in contrast to the processive DNA translocation of nucleosome remodelers, usually multi-subunit complex molecular machines that pump DNA around the histone octamer and thus slide nucleosomes and some even facilitate histone ejection and variant exchange. The resulting spaced nucleosome arrays and nucleosome-free regions are a prerequisite for DNA replication, repair and transcription. The INO80 complex is such a mega-Dalton multi-subunit nucleosome remodeler. In the third publication, we investigated the structural basis of INO80’s allosteric regulation by the so-called “A-module”. The A-module consists of nuclear actin in complex with actin-related proteins bound to a lever that feeds back to the motor ATPase. Although it is known that the Amodule binds to extranucleosomal entry DNA, we present a model that explains INO80-specificmonitoring of DNA shape by the A-module, the counter-grip subunit Arp5 and the motor ATPase itself. Consequently, mutual conformational feedback between the submodules yields a specificnucleosome positioning outcome

The COMA complex interacts with Cse4 and positions Sli15/Ipl1 at the budding yeast inner kinetochore

International audienceKinetochores are macromolecular protein complexes at centromeres that ensure accurate chromosome segregation by attaching chromosomes to spindle microtubules and integrating safeguard mechanisms. The inner kinetochore is assembled on CENP-A nucleosomes and has been implicated in establishing a kinetochore-associated pool of Aurora B kinase, a chromosomal passenger complex (CPC) subunit, which is essential for chromosome biorientation. By performing crosslink-guided in vitro reconstitution of budding yeast kinetochore complexes we showed that the Ame1/Okp1CENP-U/Q heterodimer, which forms the COMA complex with Ctf19/Mcm21CENP-P/O, selectively bound Cse4CENP-A nucleosomes through the Cse4 N-terminus. The Sli15/Ipl1INCENP/Aurora-B core-CPC interacted with COMA in vitro through the Ctf19 C-terminus whose deletion affects accurate chromosome segregation in a Sli15 wild-type background. Tethering Sli15 to Ame1/Okp1 rescued synthetic lethality upon Ctf19 depletion in a Sli15 centromere-targeting deficient mutant. This study shows molecular characteristics of the point-centromere inner kinetochore architecture and suggests a role for the Ctf19 C-terminus in mediating accurate chromosome segregation

Structural deficits in key domains of Shank2 lead to alterations in postsynaptic nanoclusters and to a neurodevelopmental disorder in humans

Postsynaptic scaffold proteins such as Shank, PSD-95, Homer and SAPAP/GKAP family members establish the postsynaptic density of glutamatergic synapses through a dense network of molecular interactions. Mutations in SHANK genes are associated with neurodevelopmental disorders including autism and intellectual disability. However, no SHANK missense mutations have been described which interfere with the key functions of Shank proteins believed to be central for synapse formation, such as GKAP binding via the PDZ domain, or Zn2+-dependent multimerization of the SAM domain. We identify two individuals with a neurodevelopmental disorder carrying de novo missense mutations in SHANK2. The p.G643R variant distorts the binding pocket for GKAP in the Shank2 PDZ domain and prevents interaction with Thr(−2) in the canonical PDZ ligand motif of GKAP. The p.L1800W variant severely delays the kinetics of Zn2+-dependent polymerization of the Shank2-SAM domain. Structural analysis shows that Trp1800 dislodges one histidine crucial for Zn2+ binding. The resulting conformational changes block the stacking of helical polymers of SAM domains into sheets through side-by-side contacts, which is a hallmark of Shank proteins, thereby disrupting the highly cooperative assembly process induced by Zn2+. Both variants reduce the postsynaptic targeting of Shank2 in primary cultured neurons and alter glutamatergic synaptic transmission. Super-resolution microscopy shows that both mutants interfere with the formation of postsynaptic nanoclusters. Our data indicate that both the PDZ- and the SAM-mediated interactions of Shank2 contribute to the compaction of postsynaptic protein complexes into nanoclusters, and that deficiencies in this process interfere with normal brain development in humans