Debugging and consolidating multiple synthetic chromosomes reveals combinatorial genetic interactions.

The Sc2.0 project is building a eukaryotic synthetic genome from scratch. A major milestone has been achieved with all individual Sc2.0 chromosomes assembled. Here, we describe the consolidation of multiple synthetic chromosomes using advanced endoreduplication intercrossing with tRNA expression cassettes to generate a strain with 6.5 synthetic chromosomes. The 3D chromosome organization and transcript isoform profiles were evaluated using Hi-C and long-read direct RNA sequencing. We developed CRISPR Directed Biallelic URA3-assisted Genome Scan, or "CRISPR D-BUGS," to map phenotypic variants caused by specific designer modifications, known as "bugs." We first fine-mapped a bug in synthetic chromosome II (synII) and then discovered a combinatorial interaction associated with synIII and synX, revealing an unexpected genetic interaction that links transcriptional regulation, inositol metabolism, and tRNASerCGA abundance. Finally, to expedite consolidation, we employed chromosome substitution to incorporate the largest chromosome (synIV), thereby consolidating >50% of the Sc2.0 genome in one strain.

A functional evolutionary approach to identify determinants of nucleosome positioning: a unifying model for establishing the genome-wide pattern.

Although the genomic pattern of nucleosome positioning is broadly conserved, quantitative aspects vary over evolutionary timescales. We identify the cis and trans determinants of nucleosome positioning using a functional evolutionary approach involving S. cerevisiae strains containing large genomic regions from other yeast species. In a foreign species, nucleosome depletion at promoters is maintained over poly(dA:dT) tracts, whereas internucleosome spacing and all other aspects of nucleosome positioning tested are not. Interestingly, the locations of the +1 nucleosome and RNA start sites shift in concert. Strikingly, in a foreign species, nucleosome-depleted regions occur fortuitously in coding regions, and they often act as promoters that are associated with a positioned nucleosome array linked to the length of the transcription unit. We suggest a three-step model in which nucleosome remodelers, general transcription factors, and the transcriptional elongation machinery are primarily involved in generating the nucleosome positioning pattern in vivo.

Consequences of a telomerase-related fitness defect and chromosome substitution technology in yeast synIX strains.

We describe the complete synthesis, assembly, debugging, and characterization of a synthetic 404,963 bp chromosome, synIX (synthetic chromosome IX). Combined chromosome construction methods were used to synthesize and integrate its left arm (synIXL) into a strain containing previously described synIXR. We identified and resolved a bug affecting expression of EST3, a crucial gene for telomerase function, producing a synIX strain with near wild-type fitness. To facilitate future synthetic chromosome consolidation and increase flexibility of chromosome transfer between distinct strains, we combined chromoduction, a method to transfer a whole chromosome between two strains, with conditional centromere destabilization to substitute a chromosome of interest for its native counterpart. Both steps of this chromosome substitution method were efficient. We observed that wild-type II tended to co-transfer with synIX and was co-destabilized with wild-type IX, suggesting a potential gene dosage compensation relationship between these chromosomes.

Transcriptional neighborhoods regulate transcript isoform lengths and expression levels.

Sequence features of genes and their flanking regulatory regions are determinants of RNA transcript isoform expression and have been used as context-independent plug-and-play modules in synthetic biology. However, genetic context-including the adjacent transcriptional environment-also influences transcript isoform expression levels and boundaries. We used synthetic yeast strains with stochastically repositioned genes to systematically disentangle the effects of sequence and context. Profiling 120 million full-length transcript molecules across 612 genomic perturbations, we observed sequence-independent alterations to gene expression levels and transcript isoform boundaries that were influenced by neighboring transcription. We identified features of transcriptional context that could predict these alterations and used these features to engineer a synthetic circuit where transcript length was controlled by neighboring transcription. This demonstrates how positional context can be leveraged in synthetic genome engineering.

Histone exchange is associated with activator function at transcribed promoters and with repression at histone loci.

Transcription in eukaryotes correlates with major chromatin changes, including the replacement of old nucleosomal histones by new histones at the promoters of genes. The role of these histone exchange events in transcription remains unclear. In particular, the causal relationship between histone exchange and activator binding, preinitiation complex (PIC) assembly, and/or subsequent transcription remains unclear. Here, we provide evidence that histone exchange at gene promoters is not simply a consequence of PIC assembly or transcription but instead is mediated by activators. We further show that not all activators up-regulate gene expression by inducing histone turnover. Thus, histone exchange does not simply correlate with transcriptional activity, but instead reflects the mode of action of the activator. Last, we show that histone turnover is not only associated with activator function but also plays a role in transcriptional repression at the histone loci.

Rehabilitation Following Fracture in Dystrophinopathy, A Case Series.

BACKGROUND: Boys with dystrophinopathies (DMD) are at increased risk of low bone mineral density and fracture. Femoral fracture is the most common extremity fracture and is accompanied by significant risk of functional loss. Care considerations for DMD have stressed that aggressive management may be needed to maintain ambulation and that surgical fixation allows early mobilization. OBJECTIVES: Describe 5 cases of femoral fracture in ambulatory boys with DMD and the course of care undertaken to optimize function. PATIENTS: Five boys with DMD median age 15y (12-16) who were independently ambulatory. Median 10m walk speed prior to their first fracture was 8 sec (7-17.37) and 4 of 5 were less than the 9 seconds predictive of 2 year ambulation retention. Three of the cases had a single incident causing fracture; the remaining cases had 2 and 3 incidents respectively representing a total of 8 fractures 6 of which were surgically stabilized. RESULTS: Following the first fracture, all 5 subjects regained some form of ambulation. Three patients regained independent ambulation and 2 with hand held support or contact guard. Two subjects went on to have additional falls with associated fracture. No patient regained the ability to rise from the floor and only one of the 5 regained the ability to climb steps and all demonstrated a decline in walking speed. CONCLUSION: Prompt orthopedic intervention, early mobility, and intensive rehabilitation even in the end stage ambulatory patient, were factors in helping preserve function in these patients with dystrophinopathies.

Structure of the chromatin remodelling enzyme Chd1 bound to a ubiquitinylated nucleosome.

ATP-dependent chromatin remodelling proteins represent a diverse family of proteins that share ATPase domains that are adapted to regulate protein-DNA interactions. Here, we present structures of the Saccharomyces cerevisiae Chd1 protein engaged with nucleosomes in the presence of the transition state mimic ADP-beryllium fluoride. The path of DNA strands through the ATPase domains indicates the presence of contacts conserved with single strand translocases and additional contacts with both strands that are unique to Snf2 related proteins. The structure provides connectivity between rearrangement of ATPase lobes to a closed, nucleotide bound state and the sensing of linker DNA. Two turns of linker DNA are prised off the surface of the histone octamer as a result of Chd1 binding, and both the histone H3 tail and ubiquitin conjugated to lysine 120 are re-orientated towards the unravelled DNA. This indicates how changes to nucleosome structure can alter the way in which histone epitopes are presented.

Deciphering Subunit-Specific Functions within SWI/SNF Complexes.

In this issue of Cell Reports, Sen et al. and Dutta et al. reveal the modularity of the yeast SWI/SNF chromatin remodeling complex and show that loss of different subunits leads to distinct consequences for gene expression.

Structural reorganization of the chromatin remodeling enzyme Chd1 upon engagement with nucleosomes.

The yeast Chd1 protein acts to position nucleosomes across genomes. Here, we model the structure of the Chd1 protein in solution and when bound to nucleosomes. In the apo state, the DNA-binding domain contacts the edge of the nucleosome while in the presence of the non-hydrolyzable ATP analog, ADP-beryllium fluoride, we observe additional interactions between the ATPase domain and the adjacent DNA gyre 1.5 helical turns from the dyad axis of symmetry. Binding in this conformation involves unravelling the outer turn of nucleosomal DNA and requires substantial reorientation of the DNA-binding domain with respect to the ATPase domains. The orientation of the DNA-binding domain is mediated by sequences in the N-terminus and mutations to this part of the protein have positive and negative effects on Chd1 activity. These observations indicate that the unfavorable alignment of C-terminal DNA-binding region in solution contributes to an auto-inhibited state.

Comparative Genomics Reveals Chd1 as a Determinant of Nucleosome Spacing in Vivo.

Packaging of genomic DNA into nucleosomes is nearly universally conserved in eukaryotes, and many features of the nucleosome landscape are quite conserved. Nonetheless, quantitative aspects of nucleosome packaging differ between species because, for example, the average length of linker DNA between nucleosomes can differ significantly even between closely related species. We recently showed that the difference in nucleosome spacing between two Hemiascomycete species-Saccharomyces cerevisiae and Kluyveromyces lactis-is established by trans-acting factors rather than being encoded in cis in the DNA sequence. Here, we generated several S. cerevisiae strains in which endogenous copies of candidate nucleosome spacing factors are deleted and replaced with the orthologous factors from K. lactis. We find no change in nucleosome spacing in such strains in which H1 or Isw1 complexes are swapped. In contrast, the K. lactis gene encoding the ATP-dependent remodeler Chd1 was found to direct longer internucleosomal spacing in S. cerevisiae, establishing that this remodeler is partially responsible for the relatively long internucleosomal spacing observed in K. lactis. By analyzing several chimeric proteins, we find that sequence differences that contribute to the spacing activity of this remodeler are dispersed throughout the coding sequence, but that the strongest spacing effect is linked to the understudied N-terminal end of Chd1. Taken together, our data find a role for sequence evolution of a chromatin remodeler in establishing quantitative aspects of the chromatin landscape in a species-specific manner.

Mechanisms underlying nucleosome positioning in vivo.

It has been some 40 years since repeating subunits in eukaryotic chromatin, initially termed "nu bodies," were described. Four decades of study have characterized the structural organization of the nucleosome, from multiple crystal structures of individual nucleosomes to genome-wide maps of nucleosome positions in scores of organisms. Nucleosome positioning can impact essentially all DNA-templated processes, making an appreciation of the forces shaping the nucleosomal landscape in eukaryotes key to fully understanding genomic regulation. Here, we review the factors impacting nucleosome positioning and the ways that nucleosomes can control the output of the genome.

Golden Gate Assembly of Transcriptional Unit Libraries into a Rearrangeable Gene Cluster.

Both regulatory sequences and genome organization contribute to the production of diverse transcript isoforms, which can influence how genes, or sets of genes, are expressed. An efficient, modular approach is needed to generate the combinatorial complexity required to empirically test many combinations of different regulatory sequences and different gene orders. Golden Gate assembly provides such a tool for seamless one-pot cleavage and ligation, by using type IIS restriction enzymes, which cleave outside of their recognition site. In addition to reducing the number of steps, this one-pot reaction can improve correct assemblies by the continued cleavage of self-ligation products that retain the recognition site. Switching the specific restriction enzyme used between steps allows for modular assembly of several units. A protocol to perform modular assemblies with two type IIS restriction enzymes, namely BsaI-v2-HF and BsmBI-v2, is described here. This protocol includes a description for generating destination vectors that add loxPsym sites between transcriptional units, allowing for diversification of gene order, orientation, and spacing.