HDAC1 overexpression independently predicts biochemical recurrence and is associated with rapid tumor cell proliferation and genomic instability in prostate cancer.

Histone deacetylases (HDACs) play an important role in tumor development and progression by modifying histone and non-histone proteins. In the current study we analyzed prevalence and prognostic impact of HDAC1 in prostate cancer. HDAC1 expression was analyzed by immunohistochemistry on a tissue microarray containing more than 12,400 prostate cancer specimens. Results were compared to tumor phenotype, biochemical recurrence, and molecular subtypes defined by ERG status as well as genomic deletions of 3p, 5q, 6q and PTEN. HDAC1 immunostaining was detectable in 75.4% of 9744 interpretable cancers and considered strong in 15.4%, moderate in 39.4% and weak in 20.7% of cases. High HDAC1 expression was associated with high Gleason grade (p<0.0001), advanced pathological tumor stage (p<0.0001), positive nodal status (p=0.0010), elevated preoperative PSA-level (p=0.0127), early PSA recurrence (p<0.0001) and increased cell proliferation (p<0.0001). Moreover, high-level HDAC1 staining was associated with TMPRSS2:ERG rearrangement and ERG expression in prostate cancers (p<0.0001) and was linked to deletions of PTEN (p<0.0001), 6q (p<0.0001) and 5q (p=0.0028) in ERG-negative cancers. The prognostic impact of HDAC1 was independent of established clinicopathological parameters and was mostly driven by ERG-negative cancers as revealed by subgroup analyses. HDAC1 has strong prognostic impact in prostate cancer and might contribute to the development of a fraction of genetically instable and particularly aggressive prostate cancers. HDAC1 measurement might therefore be of clinical value for risk stratification of prostate cancer and should be further evaluated in this regard.

A highly effective TALEN-mediated approach for targeted gene disruption in Xenopus tropicalis and zebrafish.

Transcription activator like effector nucleases (TALENs) is a promising approach to disrupt intended genomic loci. The assembly of highly effective TALENs is critical for successful genome editing. Recently we reported a convenient and robust platform to construct customized TALENs. The TALENs generated by this platform have been proven to be highly effective for gene disruption in Xenopus tropicalis and zebrafish as well as large genomic deletions in zebrafish. The one-time success rate of targeted gene disruption is about 90% for more than 100 genomic loci tested, with the mutation frequencies often reaching above 50%. Here we describe the validated protocol for TALEN assembly, methods for generating gene knockout animals in X. tropicalis and zebrafish, as well as the protocol for engineering large genomic deletions in zebrafish.

Identification of two novel large deletions in FBN1 gene by next-generation sequencing and multiplex ligation-dependent probe amplification.

BACKGROUND: Mutations in fibrillin-1 (FBN1) are known to be associated with Marfan syndrome (MFS), an autosomal dominant connective tissue disorder. Most FBN1 mutations are missense or nonsense mutations. Traditional molecular genetic testing for the FBN1 gene, like Sanger sequencing, may miss disease-causing mutations in the gene's regulatory regions or non-coding sequences, as well as partial or complete gene deletions and duplications. METHODS: Next-generation sequencing, multiplex ligation-dependent probe amplification and gap PCR were conducted on two MFS patients to screen for disease-causing mutations. RESULTS: We identified two large deletions in FBN1 from two MFS patients. One patient had a 0.23 Mb deletion (NC_000015.9:g.48550506_48779360del) including 5'UTR-exon6 of FBN1. The other patient harbored a 1416 bp deletion (NC_000015.9:g.48410869_48412284del) affecting the last exon, exon 66, of the FBN1 gene. CONCLUSION: Our results expanded the number of large FBN1 deletions and highlighted the importance of screening for large deletions in FBN1 in clinical genetic testing, especially for those with the classic MFS phenotype.

A highly efficient method for genomic deletion across diverse lengths in thermophilic Parageobacillus thermoglucosidasius.

Parageobacillus thermoglucosidasius is emerging as a highly promising thermophilic organism for metabolic engineering. The utilization of CRISPR-Cas technologies has facilitated programmable genetic manipulation in P. thermoglucosidasius. However, the absence of thermostable NHEJ enzymes limited the capability of the endogenous type I CRISPR-Cas system to generate a variety of extensive genomic deletions. Here, two thermophilic NHEJ enzymes were identified and combined with the endogenous type I CRISPR-Cas system to develop a genetic manipulation tool that can achieve long-range genomic deletion across various lengths. By optimizing this tool-through adjusting the expression level of NHEJ enzymes and leveraging our discovery of a negative correlation between GC content of the guide RNA (gRNA) and deletion efficacy-we streamlined a comprehensive gRNA selection manual for whole-genome editing, achieving a 100 % success rate in randomly selecting gRNAs. Notably, using just one gRNA, we achieved genomic deletions spanning diverse length, exceeding 200 kilobases. This tool will facilitate the genomic manipulation of P. thermoglucosidasius for both fundamental research and applied engineering studies, further unlocking its potential as a thermophilic cell factory.

Genomic deletions explain the generation of alternative BRAF isoforms conferring resistance to MAPK inhibitors in melanoma.

Resistance to MAPK inhibitors (MAPKi), the main cause of relapse in BRAF-mutant melanoma, is associated with the production of alternative BRAF mRNA isoforms (altBRAFs) in up to 30% of patients receiving BRAF inhibitor monotherapy. These altBRAFs have been described as being generated by alternative pre-mRNA splicing, and splicing modulation has been proposed as a therapeutic strategy to overcome resistance. In contrast, we report that altBRAFs are generated through genomic deletions. Using different in vitro models of altBRAF-mediated melanoma resistance, we demonstrate the production of altBRAFs exclusively from the BRAF V600E allele, correlating with corresponding genomic deletions. Genomic deletions are also detected in tumor samples from melanoma and breast cancer patients expressing altBRAFs. Along with the identification of altBRAFs in BRAF wild-type and in MAPKi-naive melanoma samples, our results represent a major shift in our understanding of mechanisms leading to the generation of BRAF transcripts variants associated with resistance in melanoma.

Phage-delivered CRISPR-Cas9 for strain-specific depletion and genomic deletions in the gut microbiome.

Mechanistic insights into the role of the human microbiome in the predisposition to and treatment of disease are limited by the lack of methods to precisely add or remove microbial strains or genes from complex communities. Here, we demonstrate that engineered bacteriophage M13 can be used to deliver DNA to Escherichia coli within the mouse gastrointestinal (GI) tract. Delivery of a programmable exogenous CRISPR-Cas9 system enables the strain-specific depletion of fluorescently marked isogenic strains during competitive colonization and genomic deletions that encompass the target gene in mice colonized with a single strain. Multiple mechanisms allow E. coli to escape targeting, including loss of the CRISPR array or even the entire CRISPR-Cas9 system. These results provide a robust and experimentally tractable platform for microbiome editing, a foundation for the refinement of this approach to increase targeting efficiency, and a proof of concept for the extension to other phage-bacterial pairs of interest.

A ceRNA approach may unveil unexpected contributors to deletion syndromes, the model of 5q- syndrome.

In genomic deletions, gene haploinsufficiency might directly configure a specific disease phenotype. Nevertheless, in some cases no functional association can be identified between haploinsufficient genes and the deletion-associated phenotype. Transcripts can act as microRNA sponges. The reduction of transcripts from the hemizygous region may increase the availability of specific microRNAs, which in turn may exert in-trans regulation of target genes outside the deleted region, eventually contributing to the phenotype. Here we prospect a competing endogenous RNA (ceRNA) approach for the identification of candidate genes target of epigenetic regulation in deletion syndromes. As a model, we analyzed the 5q- myelodysplastic syndrome. Genes in haploinsufficiency within the common 5q deleted region in CD34+ blasts were identified in silico. Using the miRWalk 2.0 platform, we predicted microRNAs whose availability, and thus activity, could be enhanced by the deletion, and performed a genomewide analysis of the genes outside the 5q deleted region that could be targeted by the predicted miRNAs. The analysis pointed to two genes with altered expression in 5q- transcriptome, which have never been related with 5q- before. The prospected approach allows investigating the global transcriptional effect of genomic deletions, possibly prompting discovery of unsuspected contributors in the deletion-associated phenotype. Moreover, it may help in functionally characterizing previously reported unexpected interactions.