Biology subjects

Lim, K.

Publications and source records attributed to Lim, K..

44 records · Page 3Linked to original sources

bioRxiv2020

Reprogramming of the FOXA1 cistrome in treatment-emergent neuroendocrine prostate cancer

Lineage plasticity, the ability of a cell to alter its identity, is an increasingly common mechanism of adaptive resistance to targeted therapy in cancer1,2. An archetypal example is the development of neuroendocrine prostate cancer (NEPC) after treatment of prostate adenocarcinoma (PRAD) with inhibitors of androgen signaling. NEPC is an aggressive variant of prostate cancer that aberrantly expresses genes characteristic of neuroendocrine (NE) tissues and no longer depends on androgens. To investigate the epigenomic basis of this resistance mechanism, we profiled histone modifications in NEPC and PRAD patient-derived xenografts (PDXs) using chromatin immunoprecipitation and sequencing (ChIP-seq). We identified a vast network of cis-regulatory elements (N~15,000) that are recurrently activated in NEPC. The FOXA1 transcription factor (TF), which pioneers androgen receptor (AR) chromatin binding in the prostate epithelium3,4, is reprogrammed to NE-specific regulatory elements in NEPC. Despite loss of dependence upon AR, NEPC maintains FOXA1 expression and requires FOXA1 for proliferation and expression of NE lineage-defining genes. Ectopic expression of the NE lineage TFs ASCL1 and NKX2-1 in PRAD cells reprograms FOXA1 to bind to NE regulatory elements and induces enhancer activity as evidenced by histone modifications at these sites. Our data establish the importance of FOXA1 in NEPC and provide a principled approach to identifying novel cancer dependencies through epigenomic profiling.

cancer biology↗

bioRxiv2020

Subtype Heterogeneity and Epigenetic Convergence in Neuroendocrine Prostate Cancer

Neuroendocrine carcinomas (NEC) are tumors expressing markers of neuronal differentiation that can arise at different anatomic sites but have strong histological and clinical similarities. Here we report the chromatin landscapes of a range of human NECs and show convergence to the activation of a common epigenetic program. With a particular focus on treatment emergent neuroendocrine prostate cancer (NEPC), we analyzed cell lines, patient-derived xenograft (PDX) models and human clinical samples to show the existence of two distinct NEPC subtypes based on the expression of the neuronal transcription factors ASCL1 and NEUROD1. While in cell lines and PDX models these subtypes are mutually exclusive, single cell analysis of human clinical samples exhibit a more complex tumor structure with subtypes coexisting as separate sub-populations within the same tumor. These tumor sub-populations differ genetically and epigenetically contributing to intra- and inter-tumoral heterogeneity in human metastases. Overall our results provide a deeper understanding of the shared clinicopathological characteristics shown by NECs. Furthermore, the intratumoral heterogeneity of human NEPCs suggests the requirement of simultaneous targeting of coexisting tumor populations as a therapeutic strategy.

cancer biology↗

bioRxiv2020

Phylotype-Level Characterization of Complex Lactobacilli Communities Using a High-Throughput, High-Resolution Phenylalanyl-tRNA Synthetase (pheS) Gene Amplicon Sequencing Approach

The lactobacilli to date encompass more than 270 closely related species that were recently re-classified into 26 genera. Because of their relevance to industry, there is a need to distinguish between closely related, yet metabolically and regulatory distinct species, e.g., during monitoring of biotechnological processes or screening of samples of unknown composition. Current available methods, such as shotgun metagenomics or rRNA-based amplicon sequencing have significant limitations (high cost, low resolution, etc.). Here, we generated a lactobacilli phylogeny based on phenylalanyl-tRNA synthetase (pheS) genes and, from it, developed a high-resolution taxonomic framework which allows for comprehensive and confident characterization of lactobacilli community diversity and structure at the species-level. This framework is based on a total of 445 pheS gene sequences, including sequences of 277 validly described species and subspecies (out of a total of 283, coverage of 98%). It allows differentiation between 263 lactobacilli species-level clades out of a total of 273 validly described species (including the proposed species L. timonensis) and a further two subspecies. The methodology was validated through next-generation sequencing of mock communities. At a sequencing depth of [~]30,000 sequences, the minimum level of detection was approximately 0.02 pg per l DNA (equalling approximately 10 genome copies per {micro}l template DNA). The pheS approach along with parallel sequencing of partial 16S rRNA genes revealed a considerable lactobacilli diversity and distinct community structures across a broad range of samples from different environmental niches. This novel complementary approach may be applicable to industry and academia alike. IMPORTANCESpecies within the former genera Lactobacillus and Pediococcus have been studied extensively at the genomic level. To accommodate for their exceptional functional diversity, the over 270 species were recently re-classified into 26 distinct genera. Despite their relevance to both academia and industry, methods that allow detailed exploration of their ecology are still limited by low resolution, high cost or copy number variations. The approach described here makes use of a single copy marker gene which outperforms other markers with regards to species-level resolution and availability of reference sequences (98% coverage). The tool was validated against a mock community and used to address lactobacilli diversity and community structure in various environmental matrices. Such analyses can now be performed at broader scale to assess and monitor lactobacilli community assembly, structure and function at the species (in some cases even at sub-species) level across a wide range of academic and commercial applications.

microbiology↗

bioRxiv2020

The cryo-electron microscopy structure of the human CDK-activating kinase

The human CDK-activating kinase (CAK), a complex composed of cyclin dependent kinase (CDK) 7, cyclin H, and MAT1, is a critical regulator of transcription initiation and the cell cycle. It acts by phosphorylating the C-terminal heptapeptide repeat domain of the RNA polymerase II subunit Rpb1, which is an important regulatory event in transcription initiation by Pol II, and it phosphorylates the regulatory T-loop of CDKs that control cell-cycle progression. Here, we have determined the three-dimensional structure of the catalytic module of human CAK, revealing the structural basis of its assembly and providing insight into CDK7 activation in this context. The unique third component of the complex, MAT1, substantially extends the interaction interface between CDK7 and cyclin H, explaining its role as a CAK assembly factor, and it forms interactions with the CDK7 T-loop, which may contribute to enhancing CAK activity. We have also determined the structure of the CAK in complex with the covalently bound inhibitor THZ1 in order to provide insight into the binding of inhibitors at the CDK7 active site and aid in the rational design of therapeutic compounds. SignificanceControl of gene expression and the cell cycle is critical for appropriate cell growth and timely cell division. Failure of the mechanisms regulating these processes can result in proliferative diseases. A molecular complex termed the CDK activating kinase (CAK) impinges on both of these regulatory networks in human cells and is thus a possible drug target for treatment of cancer. Here, we use cryo-electron microscopy to describe the detailed molecular structure of the human CAK, revealing its architecture and the interactions between its regulatory elements. Additionally, we have obtained the structure of the CAK in complex with a small-molecule inhibitor.

biochemistry↗

bioRxiv2018

Genome Wide Study of Tardive Dyskinesia in Schizophrenia

Tardive dyskinesia (TD) is a severe condition characterized by repetitive involuntary movement of orofacial regions and extremities. Patients treated with antipsychotics typically present with TD symptomatology. Here, we conducted the largest GWAS of TD to date, by meta-analyzing samples of East-Asian, European, and African-American ancestry, followed by analyses of biological pathways and polygenic risk with related phenotypes. We identified a novel locus and three suggestive loci, implicating immune-related pathways. Through integrating trans-ethnic fine-mapping, we identified putative credible causal variants for three of the loci. Multivariate analyses of polygenic risk for TD supports the genetic susceptibility of TD, with relatively lower allele frequencies variants being associated with TD, beyond that of antipsychotic medication. Together, these findings provide new insights into the genetic architecture and biology of TD.

genomics↗

bioRxiv2018

Web-based design and analysis tools for CRISPR base editing

BackgroundAs a result of its simplicity and high efficiency, the CRISPR-Cas system has been widely used as a genome editing tool. Recently, CRISPR base editors, which consist of deactivated Cas9 (dCas9) or Cas9 nickase (nCas9) linked with a cytidine or a guanine deaminase, have been developed. Base editing tools will be very useful for gene correction because they can produce highly specific DNA substitutions without the introduction of any donor DNA, but dedicated web-based tools to facilitate the use of such tools have not yet been developed.\n\nResultsWe present two web tools for base editors, named BE-Designer and BE-Analyzer. BE-Designer provides all possible base editor target sequences in a given input DNA sequence with useful information including potential off-target sites. BE-Analyzer, a tool for assessing base editing outcomes from next generation sequencing (NGS) data, provides information about mutations in a table and interactive graphs. Furthermore, because the tool runs client-side, large amounts of targeted deep sequencing data (>100MB) do not need to be uploaded to a server, substantially reducing running time and increasing data security. BE-Designer and BE-Analyzer can be freely accessed at http://www.rgenome.net/bedesigner/ and http://www.rgenome.net/be-analyzer/respectively\n\nConclusionWe develop two useful web tools to design target sequence (BE-Designer) and to analyze NGS data from experimental results (BE-Analyzer) for CRISPR base editors.

bioinformatics↗

bioRxiv2018

Genome-wide scan and fine-mapping of rare nonsynonymous associations implicates intracellular lipolysis genes in fat distribution and cardio-metabolic risk

Difficulties in identifying causal variants and genes underlying genetic associations have limited the translational potential of genetic studies of body fat distribution, an important, partly-heritable risk factor for cardio-metabolic disease. Rare variant associations facilitate fine-mapping of causal alleles, but their contribution to fat distribution is understudied. We performed a genome-wide scan of rare nonsynonymous variants for body mass index-adjusted waist-to-hip-ratio (BMI-adjusted WHR; a widely-used measure of fat distribution) in 450,562 European ancestry individuals, followed by systematic Bayesian fine-mapping at six genome-wide (p<5x10-08; main-analysis) and two subthreshold signals (significant at a Bonferroni-corrected p<1.3x10-06). We found strong statistical evidence of causal association for nonsynonymous alleles in CALCRL (p.L87P, pconditional=5.9x10-12; posterior-probability of association [PPA]=52%), PLIN1 (p.L90P, pconditional=5.5x10-13; PPA>99%), PDE3B (p.R783X, pconditional=6.2x10-15; PPA>99%), ACVR1C (p.I195T; pconditional=5.4x10-12; PPA>99%), and FGF1 (p.G21E, pconditional=1.6x10-07; PPA=98%). Alleles at the four likely-causal main-analysis genes affected fat distribution primarily via larger hip-rather than smaller waist-circumference and six of nine conditionally-independent WHR-lowering index-variants were associated with protection from cardiovascular or metabolic disease. All four genes are expressed in adipose tissue and have been linked with the regulation of intracellular lipolysis, which controls fat retention in mature cells. Targeted follow-up analyses of key intracellular-lipolysis genes revealed associations for a variant in the initiator of intracellular lipolysis PNPLA2 (p.N252K) with higher BMI-adjusted-WHR and higher cardio-metabolic risk. This study provides human genetic evidence of a link between intracellular lipolysis, fat-distribution and its cardio-metabolic complications in the general population.

genetics↗

bioRxiv2016

Simple adjustment of the sequence weight algorithm remarkably enhances PSI-BLAST performance

PSI-BLAST, an extremely popular tool for sequence similarity search, features the utilization of Position Specific Scoring Matrix (PSSM) constructed from a multiple sequence alignment (MSA). PSSM allows the detection of more distant homologs than a general amino acid substitution matrix does. An accurate estimation of the weights of sequences in an MSA is crucially important for PSSM construction. PSI-BLAST divides a given MSA into multiple blocks, for which sequence weights are calculated. When the block width becomes very narrow, the sequence weight calculation can be difficult.\n\nWe demonstrate that PSI-BLAST indeed generates a significant fraction of blocks having widths less than 5, thereby degrading the PSI-BLAST performance. We revised the code of PSI-BLAST to prevent the blocks from being narrower than a given minimum block width (MBW). We designate the modified application of PSI-BLAST as PSI-BLASTexB. When MBW is 25, PSI-BLASTexB notably outperforms PSI-BLAST consistently for three independent benchmark sets. The performance boost is even more drastic when an MSA, instead of a sequence, was used as a query.\n\nOur results demonstrate that the generation of narrow-width blocks during the sequence weight calculation is a critically important factor that restricts the PSI-BLAST search performance. By preventing narrow blocks, PSI-BLASTexB remarkably upgrades the PSI-BLAST performance.

bioinformatics↗