Human TSC2 Mutant Cells Exhibit Aberrations in Early Neurodevelopment Accompanied by Changes in the DNA Methylome.

Tuberous Sclerosis Complex (TSC) is a debilitating developmental disorder characterized by a variety of clinical manifestations. While benign tumors in the heart, lungs, kidney, and brain are all hallmarks of the disease, the most severe symptoms of TSC are often neurological, including seizures, autism, psychiatric disorders, and intellectual disabilities. TSC is caused by loss of function mutations in the TSC1 or TSC2 genes and consequent dysregulation of signaling via mechanistic Target of Rapamycin Complex 1 (mTORC1). While TSC neurological phenotypes are well-documented, it is not yet known how early in neural development TSC1/2-mutant cells diverge from the typical developmental trajectory. Another outstanding question is the contribution of homozygous-mutant cells to disease phenotypes and whether such phenotypes are also seen in the heterozygous-mutant populations that comprise the vast majority of cells in patients. Using TSC patient-derived isogenic induced pluripotent stem cells (iPSCs) with defined genetic changes, we observed aberrant early neurodevelopment in vitro, including misexpression of key proteins associated with lineage commitment and premature electrical activity. These alterations in differentiation were coincident with hundreds of differentially methylated DNA regions, including loci associated with key genes in neurodevelopment. Collectively, these data suggest that mutation or loss of TSC2 affects gene regulation and expression at earlier timepoints than previously appreciated, with implications for whether and how prenatal treatment should be pursued.

Genomic dissection and mutation-specific target discovery for breast cancer PIK3CA hotspot mutations.

BACKGROUND: Recent advancements in high-throughput genomics and targeted therapies have provided tremendous potential to identify and therapeutically target distinct mutations associated with cancers. However, to date the majority of targeted therapies are used to treat all functional mutations within the same gene, regardless of affected codon or phenotype. RESULTS: In this study, we developed a functional genomic analysis workflow with a unique isogenic cell line panel bearing two distinct hotspot PIK3CA mutations, E545K and H1047R, to accurately identify targetable differences between mutations within the same gene. We performed RNA-seq and ATAC-seq and identified distinct transcriptomic and epigenomic differences associated with each PIK3CA hotspot mutation. We used this data to curate a select CRISPR knock out screen to identify mutation-specific gene pathway vulnerabilities. These data revealed AREG as a E545K-preferential target that was further validated through in vitro analysis and publicly available patient databases. CONCLUSIONS: Using our multi-modal genomics framework, we discover distinct differences in genomic regulation between PIK3CA hotspot mutations, suggesting the PIK3CA mutations have different regulatory effects on the function and downstream signaling of the PI3K complex. Our results demonstrate the potential to rapidly uncover mutation specific molecular targets, specifically AREG and a proximal gene regulatory region, that may provide clinically relevant therapeutic targets. The methods outlined provide investigators with an integrative strategy to identify mutation-specific targets for the treatment of other oncogenic mutations in an isogenic system.

Enhancer-promoter activation by the Kaposi sarcoma-associated herpesvirus episome maintenance protein LANA.

Higher-order genome structure influences the transcriptional regulation of cellular genes through the juxtaposition of regulatory elements, such as enhancers, close to promoters of target genes. While enhancer activation has emerged as an important facet of Kaposi sarcoma-associated herpesvirus (KSHV) biology, the mechanisms controlling enhancer-target gene expression remain obscure. Here, we discover that the KSHV genome tethering protein latency-associated nuclear antigen (LANA) potentiates enhancer-target gene expression in primary effusion lymphoma (PEL), a highly aggressive B cell lymphoma causally associated with KSHV. Genome-wide analyses demonstrate increased levels of enhancer RNA transcription as well as activating chromatin marks at LANA-bound enhancers. 3D genome conformation analyses identified genes critical for latency and tumorigenesis as targets of LANA-occupied enhancers, and LANA depletion results in their downregulation. These findings reveal a mechanism in enhancer-gene coordination and describe a role through which the main KSHV tethering protein regulates essential gene expression in PEL.

Genomic dissection and mutation-specific target discovery for breast cancer PIK3CA hotspot mutations.

Background: Recent advancements in high-throughput genomics and targeted therapies have provided tremendous potential to identify and therapeutically target distinct mutations associated with cancers. However, to date the majority of targeted therapies are used to treat all functional mutations within the same gene, regardless of affected codon or phenotype. Results: In this study, we developed a functional genomic analysis workflow with a unique isogenic cell line panel bearing two distinct hotspot PIK3CA mutations, E545K and H1047R, to accurately identify targetable differences between mutations within the same gene. We performed RNA-seq and ATAC-seq and identified distinct transcriptomic and epigenomic differences associated with each PIK3CA hotspot mutation. We used this data to curate a select CRISPR knock out screen to identify mutation-specific gene pathway vulnerabilities. These data revealed AREG as a E545K-preferential target that was further validated through in vitro analysis and publicly available patient databases. Conclusions: Using our multi-modal genomics framework, we discover distinct differences in genomic regulation between PIK3CA hotspot mutations, suggesting the PIK3CA mutations have different regulatory effects on the function and downstream signaling of the PI3K complex. Our results demonstrate the potential to rapidly uncover mutation specific molecular targets, specifically AREG and a proximal gene regulatory region, that may provide clinically relevant therapeutic targets. The methods outlined provide investigators with an integrative strategy to identify mutation-specific targets for the treatment of other oncogenic mutations in an isogenic system.

Cross-tissue patterns of DNA hypomethylation reveal genetically distinct histories of cell development.

BACKGROUND: Establishment of DNA methylation (DNAme) patterns is essential for balanced multi-lineage cellular differentiation, but exactly how these patterns drive cellular phenotypes is unclear. While > 80% of CpG sites are stably methylated, tens of thousands of discrete CpG loci form hypomethylated regions (HMRs). Because they lack DNAme, HMRs are considered transcriptionally permissive, but not all HMRs actively regulate genes. Unlike promoter HMRs, a subset of non-coding HMRs is cell type-specific and enriched for tissue-specific gene regulatory functions. Our data further argues not only that HMR establishment is an important step in enforcing cell identity, but also that cross-cell type and spatial HMR patterns are functionally informative of gene regulation. RESULTS: To understand the significance of non-coding HMRs, we systematically dissected HMR patterns across diverse human cell types and developmental timepoints, including embryonic, fetal, and adult tissues. Unsupervised clustering of 126,104 distinct HMRs revealed that levels of HMR specificity reflects a developmental hierarchy supported by enrichment of stage-specific transcription factors and gene ontologies. Using a pseudo-time course of development from embryonic stem cells to adult stem and mature hematopoietic cells, we find that most HMRs observed in differentiated cells (~ 60%) are established at early developmental stages and accumulate as development progresses. HMRs that arise during differentiation frequently (~ 35%) establish near existing HMRs (≤ 6 kb away), leading to the formation of HMR clusters associated with stronger enhancer activity. Using SNP-based partitioned heritability from GWAS summary statistics across diverse traits and clinical lab values, we discovered that genetic contribution to trait heritability is enriched within HMRs. Moreover, the contribution of heritability to cell-relevant traits increases with both increasing HMR specificity and HMR clustering, supporting the role of distinct HMR subsets in regulating normal cell function. CONCLUSIONS: Our results demonstrate that the entire HMR repertoire within a cell-type, rather than just the cell type-specific HMRs, stores information that is key to understanding and predicting cellular phenotypes. Ultimately, these data provide novel insights into how DNA hypo-methylation provides genetically distinct historical records of a cell's journey through development, highlighting HMRs as functionally distinct from other epigenomic annotations.

Human SMARCA5 is continuously required to maintain nucleosome spacing.

Genetic models suggested that SMARCA5 was required for DNA-templated events including transcription, DNA replication, and DNA repair. We engineered a degron tag into the endogenous alleles of SMARCA5, a catalytic component of the imitation switch complexes in three different human cell lines to define the effects of rapid degradation of this key regulator. Degradation of SMARCA5 was associated with a rapid increase in global nucleosome repeat length, which may allow greater chromatin compaction. However, there were few changes in nascent transcription within the first 6 h of degradation. Nevertheless, we demonstrated a requirement for SMARCA5 to control nucleosome repeat length at G1/S and during the S phase. SMARCA5 co-localized with CTCF and H2A.Z, and we found a rapid loss of CTCF DNA binding and disruption of nucleosomal phasing around CTCF binding sites. This spatiotemporal analysis indicates that SMARCA5 is continuously required for maintaining nucleosomal spacing.

Polycomb repressor complex 2 suppresses interferon-responsive MHC-II expression in melanoma cells and is associated with anti-PD-1 resistance.

BACKGROUND: Despite the remarkable success of immunotherapy in treating melanoma, understanding of the underlying mechanisms of resistance remains limited. Emerging evidence suggests that upregulation of tumor-specific major histocompatibility complex-II (tsMHC-II) serves as a predictive marker for the response to anti-programmed death-1 (PD-1)/programmed death ligand 1 (PD-L1) therapy in various cancer types. The genetic and epigenetic pathways modulating tsMHC-II expression remain incompletely characterized. Here, we provide evidence that polycomb repressive complex 2 (PRC2)/EZH2 signaling and resulting H3K27 hypermethylation suppresses tsMHC-II. METHODS: RNA sequencing data from tumor biopsies from patients with cutaneous melanoma treated with or without anti-PD-1, targeted inhibition assays, and assays for transposase-accessible chromatin with sequencing were used to observe the relationship between EZH2 inhibition and interferon (IFN)-γ inducibility within the MHC-II pathway. RESULTS: We find that increased EZH2 pathway messenger RNA (mRNA) expression correlates with reduced mRNA expression of both presentation and T-cell genes. Notably, targeted inhibition assays revealed that inhibition of EZH2 influences the expression dynamics and inducibility of the MHC-II pathway following IFN-γ stimulation. Additionally, our analysis of patients with metastatic melanoma revealed a significant inverse association between PRC2-related gene expression and response to anti-PD-1 therapy. CONCLUSIONS: Collectively, our findings demonstrate that EZH2 inhibition leads to enhanced MHC-II expression potentially resulting from improved chromatin accessibility at CIITA, the master regulator of MHC-II. These insights shed light on the molecular mechanisms involved in tsMHC-II suppression and highlight the potential of targeting EZH2 as a therapeutic strategy to improve immunotherapy efficacy.

SLC5A3-Dependent Myo-inositol Auxotrophy in Acute Myeloid Leukemia.

An enhanced requirement for nutrients is a hallmark property of cancer cells. Here, we optimized an in vivo genetic screening strategy in acute myeloid leukemia (AML), which led to the identification of the myo-inositol transporter SLC5A3 as a dependency in this disease. We demonstrate that SLC5A3 is essential to support a myo-inositol auxotrophy in AML. The commonality among SLC5A3-dependent AML lines is the transcriptional silencing of ISYNA1, which encodes the rate-limiting enzyme for myo-inositol biosynthesis, inositol-3-phosphate synthase 1. We use gain- and loss-of-function experiments to reveal a synthetic lethal genetic interaction between ISYNA1 and SLC5A3 in AML, which function redundantly to sustain intracellular myo-inositol. Transcriptional silencing and DNA hypermethylation of ISYNA1 occur in a recurrent manner in human AML patient samples, in association with IDH1/IDH2 and CEBPA mutations. Our findings reveal myo-inositol as a nutrient dependency in AML caused by the aberrant silencing of a biosynthetic enzyme. SIGNIFICANCE: We show how epigenetic silencing can provoke a nutrient dependency in AML by exploiting a synthetic lethality relationship between biosynthesis and transport of myo-inositol. Blocking the function of this solute carrier may have therapeutic potential in an epigenetically defined subset of AML.This article is highlighted in the In This Issue feature, p. 275.

Dual detection of chromatin accessibility and DNA methylation using ATAC-Me.

The epigenome is multidimensional, with individual molecular components operating on different levels to control transcriptional output. Techniques that combine measurements of these features can reveal their precise correspondence in genomic space, or temporal connectivity, to better understand how they jointly regulate genes. ATAC-Me is an integrated method to probe DNA methylation and chromatin accessibility from a single DNA fragment library. Intact nuclei undergo Tn5 transposition to isolate DNA fragments within nucleosome-free regions. Isolated fragments are exposed to sodium bisulfite before library amplification and sequencing. A typical ATAC-Me experiment detects ~60,000-75,000 peak regions (P < 0.05), covering ~3-4 million CpG sites with at least 5× coverage. These sites display a range of methylation values depending on the cellular and genomic context. The approach is well suited for time course studies that aim to capture chromatin and DNA methylation dynamics in tandem during cellular differentiation. The protocol is completed in 2 d with standard molecular biology equipment and expertise. Analysis of resulting data uses publicly available software requiring basic bioinformatics skills to interpret results.

Transcriptional Silencing of ALDH2 Confers a Dependency on Fanconi Anemia Proteins in Acute Myeloid Leukemia.

Hundreds of genes become aberrantly silenced in acute myeloid leukemia (AML), with most of these epigenetic changes being of unknown functional consequence. Here, we demonstrate how gene silencing can lead to an acquired dependency on the DNA repair machinery in AML. We make this observation by profiling the essentiality of the ubiquitination machinery in cancer cell lines using domain-focused CRISPR screening, which revealed Fanconi anemia (FA) proteins UBE2T and FANCL as unique dependencies in AML. We demonstrate that these dependencies are due to a synthetic lethal interaction between FA proteins and aldehyde dehydrogenase 2 (ALDH2), which function in parallel pathways to counteract the genotoxicity of endogenous aldehydes. We show DNA hypermethylation and silencing of ALDH2 occur in a recurrent manner in human AML, which is sufficient to confer FA pathway dependency. Our study suggests that targeting of the ubiquitination reaction catalyzed by FA proteins can eliminate ALDH2-deficient AML. SIGNIFICANCE: Aberrant gene silencing is an epigenetic hallmark of human cancer, but the functional consequences of this process are largely unknown. In this study, we show how an epigenetic alteration leads to an actionable dependency on a DNA repair pathway through the disabling of genetic redundancy.This article is highlighted in the In This Issue feature, p. 2113.

ATAC-Me Captures Prolonged DNA Methylation of Dynamic Chromatin Accessibility Loci during Cell Fate Transitions.

DNA methylation of enhancers is dynamic, cell-type specific, and vital for cell fate progression. However, current models inadequately define its role within the hierarchy of gene regulation. Analysis of independent datasets shows an unanticipated overlap between DNA methylation and chromatin accessibility at enhancers of steady-state stem cells, suggesting that these two opposing features might exist concurrently. To define their temporal relationship, we developed ATAC-Me, which probes accessibility and methylation from single DNA library preparations. We identified waves of accessibility occurring rapidly across thousands of myeloid enhancers in a monocyte-to-macrophage cell fate model. Prolonged methylation states were observed at a majority of these sites, while transcription of nearby genes tracked closely with accessibility. ATAC-Me uncovers a significant disconnect between chromatin accessibility, DNA methylation status, and gene activity. This unexpected observation highlights the value of ATAC-Me in constructing precise molecular timelines for understanding the role of DNA methylation in gene regulation.

Mutational landscape of EGFR-, MYC-, and Kras-driven genetically engineered mouse models of lung adenocarcinoma.

Genetically engineered mouse models (GEMMs) of cancer are increasingly being used to assess putative driver mutations identified by large-scale sequencing of human cancer genomes. To accurately interpret experiments that introduce additional mutations, an understanding of the somatic genetic profile and evolution of GEMM tumors is necessary. Here, we performed whole-exome sequencing of tumors from three GEMMs of lung adenocarcinoma driven by mutant epidermal growth factor receptor (EGFR), mutant Kirsten rat sarcoma viral oncogene homolog (Kras), or overexpression of MYC proto-oncogene. Tumors from EGFR- and Kras-driven models exhibited, respectively, 0.02 and 0.07 nonsynonymous mutations per megabase, a dramatically lower average mutational frequency than observed in human lung adenocarcinomas. Tumors from models driven by strong cancer drivers (mutant EGFR and Kras) harbored few mutations in known cancer genes, whereas tumors driven by MYC, a weaker initiating oncogene in the murine lung, acquired recurrent clonal oncogenic Kras mutations. In addition, although EGFR- and Kras-driven models both exhibited recurrent whole-chromosome DNA copy number alterations, the specific chromosomes altered by gain or loss were different in each model. These data demonstrate that GEMM tumors exhibit relatively simple somatic genotypes compared with human cancers of a similar type, making these autochthonous model systems useful for additive engineering approaches to assess the potential of novel mutations on tumorigenesis, cancer progression, and drug sensitivity.

De novo DNA demethylation and noncoding transcription define active intergenic regulatory elements.

Deep sequencing of mammalian DNA methylomes has uncovered a previously unpredicted number of discrete hypomethylated regions in intergenic space (iHMRs). Here, we combined whole-genome bisulfite sequencing data with extensive gene expression and chromatin-state data to define functional classes of iHMRs, and to reconstruct the dynamics of their establishment in a developmental setting. Comparing HMR profiles in embryonic stem and primary blood cells, we show that iHMRs mark an exclusive subset of active DNase hypersensitive sites (DHS), and that both developmentally constitutive and cell-type-specific iHMRs display chromatin states typical of distinct regulatory elements. We also observe that iHMR changes are more predictive of nearby gene activity than the promoter HMR itself, and that expression of noncoding RNAs within the iHMR accompanies full activation and complete demethylation of mature B cell enhancers. Conserved sequence features corresponding to iHMR transcript start sites, including a discernible TATA motif, suggest a conserved, functional role for transcription in these regions. Similarly, we explored both primate-specific and human population variation at iHMRs, finding that while enhancer iHMRs are more variable in sequence and methylation status than any other functional class, conservation of the TATA box is highly predictive of iHMR maintenance, reflecting the impact of sequence plasticity and transcriptional signals on iHMR establishment. Overall, our analysis allowed us to construct a three-step timeline in which (1) intergenic DHS are pre-established in the stem cell, (2) partial demethylation of blood-specific intergenic DHSs occurs in blood progenitors, and (3) complete iHMR formation and transcription coincide with enhancer activation in lymphoid-specified cells.

Directional DNA methylation changes and complex intermediate states accompany lineage specificity in the adult hematopoietic compartment.

DNA methylation has been implicated as an epigenetic component of mechanisms that stabilize cell-fate decisions. Here, we have characterized the methylomes of human female hematopoietic stem/progenitor cells (HSPCs) and mature cells from the myeloid and lymphoid lineages. Hypomethylated regions (HMRs) associated with lineage-specific genes were often methylated in the opposing lineage. In HSPCs, these sites tended to show intermediate, complex patterns that resolve to uniformity upon differentiation, by increased or decreased methylation. Promoter HMRs shared across diverse cell types typically display a constitutive core that expands and contracts in a lineage-specific manner to fine-tune the expression of associated genes. Many newly identified intergenic HMRs, both constitutive and lineage specific, were enriched for factor binding sites with an implied role in genome organization and regulation of gene expression, respectively. Overall, our studies represent an important reference data set and provide insights into directional changes in DNA methylation as cells adopt terminal fates.

Deep sequencing of target linkage assay-identified regions in familial breast cancer: methods, analysis pipeline and troubleshooting.

BACKGROUND: The classical candidate-gene approach has failed to identify novel breast cancer susceptibility genes. Nowadays, massive parallel sequencing technology allows the development of studies unaffordable a few years ago. However, analysis protocols are not yet sufficiently developed to extract all information from the huge amount of data obtained. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we performed high throughput sequencing in two regions located on chromosomes 3 and 6, recently identified by linkage studies by our group as candidate regions for harbouring breast cancer susceptibility genes. In order to enrich for the coding regions of all described genes located in both candidate regions, a hybrid-selection method on tiling microarrays was performed. CONCLUSIONS/SIGNIFICANCE: We developed an analysis pipeline based on SOAP aligner to identify candidate variants with a high real positive confirmation rate (0.89), with which we identified eight variants considered candidates for functional studies. The results suggest that the present strategy might be a valid second step for identifying high penetrance genes.

High definition profiling of mammalian DNA methylation by array capture and single molecule bisulfite sequencing.

DNA methylation stabilizes developmentally programmed gene expression states. Aberrant methylation is associated with disease progression and is a common feature of cancer genomes. Presently, few methods enable quantitative, large-scale, single-base resolution mapping of DNA methylation states in desired regions of a complex mammalian genome. Here, we present an approach that combines array-based hybrid selection and massively parallel bisulfite sequencing to profile DNA methylation in genomic regions spanning hundreds of thousands of bases. This single molecule strategy enables methylation variable positions to be quantitatively examined with high sampling precision. Using bisulfite capture, we assessed methylation patterns across 324 randomly selected CpG islands (CGI) representing more than 25,000 CpG sites. A single lane of Illumina sequencing permitted methylation states to be definitively called for >90% of target sties. The accuracy of the hybrid-selection approach was verified using conventional bisulfite capillary sequencing of cloned PCR products amplified from a subset of the selected regions. This confirmed that even partially methylated states could be successfully called. A comparison of human primary and cancer cells revealed multiple differentially methylated regions. More than 25% of islands showed complex methylation patterns either with partial methylation states defining the entire CGI or with contrasting methylation states appearing in specific regional blocks within the island. We observed that transitions in methylation state often correlate with genomic landmarks, including transcriptional start sites and intron-exon junctions. Methylation, along with specific histone marks, was enriched in exonic regions, suggesting that chromatin states can foreshadow the content of mature mRNAs.

Sorting of small RNAs into Arabidopsis argonaute complexes is directed by the 5' terminal nucleotide.

Argonaute (AGO) proteins recruit small RNAs to form the core of RNAi effector complexes. Arabidopsis encodes ten AGO proteins and a large network of small RNAs. How these small RNAs are sorted into specific AGO complexes remains largely unknown. We have cataloged small RNAs resident in four AGO complexes. We found that AGO2 and AGO4 preferentially recruit small RNAs with a 5' terminal adenosine, whereas AGO1 harbors microRNAs (miRNAs) that favor a 5' terminal uridine. AGO5 predominantly binds small RNAs that initiate with cytosine. Changing the 5' terminal nucleotide of an miRNA predictably redirected it into a different AGO complex and alters its biological activity. These results reveal a role for small RNA sequences in assorting among AGO complexes. This suggests that specialization of AGO complexes might involve remodeling the 5' end-binding pocket to accept certain small RNA sequences, perhaps explaining the evolutionary drive for miRNAs to initiate with uridine.

Thorough validation of siRNA-induced cell death phenotypes defines new anti-apoptotic protein.

Loss-of-function by means of RNA interference in cultured human cells enables rapid pathway dissection on a genome-scale. Improved siRNA design and key validation protocols are required to eliminate falsely identified phenotypes resulting from potential off-target consequences. Here, we demonstrate a validation strategy involving several steps for verifying cell death phenotypes revealed during loss-of-function screening. First, from a set of 45 novel human genes we identified gene candidates that, when silenced, induce apoptosis in cultured HeLa cells. For those candidates, we performed more extensive validation with multiple effective siRNAs. In addition, we designed rescue experiments involving candidate genes delivered exogenously and containing silent mutations in the siRNA target regions. Rescue of the observed knockdown phenotype demonstrated an original and more stringent validation of the siRNA's selectivity and the phenotype specificity for the target gene. As a result, our data reveals an anti-apoptotic function for novel human breast adenocarcinoma marker BC-2, adding new depth to BC-2's description as a putative tumor marker involved in cancer related pathways.

Accelerated discovery of novel protein function in cultured human cells.

Experimental approaches that enable direct investigation of human protein function are necessary for comprehensive annotation of the human proteome. We introduce a cell-based platform for rapid and unbiased functional annotation of undercharacterized human proteins. Utilizing a library of antibody biomarkers, the full-length proteins are investigated by tracking phenotypic changes caused by overexpression in human cell lines. We combine reverse transfection and immunodetection by fluorescence microscopy to facilitate this procedure at high resolution. Demonstrating the advantage of this approach, new annotations are provided for two novel proteins: 1) a membrane-bound O-acyltransferase protein (C3F) that, when overexpressed, disrupts Golgi and endosome integrity due likely to an endoplasmic reticulum-Golgi transport block and 2) a tumor marker (BC-2) that prompts a redistribution of a transcriptional silencing protein (BMI1) and a mitogen-activated protein kinase mediator (Rac1) to distinct nuclear regions that undergo chromatin compaction. Our strategy is an immediate application for directly addressing those proteins whose molecular function remains unknown.

A combined approach exploring gene function based on worm-human orthology.

BACKGROUND: Many aspects of the nematode Caenorhabditis elegans biology are conserved between invertebrates and vertebrates establishing this particular organism as an excellent genetic model. Because of its small size, large populations and self-fertilization of the hermaphrodite, functional predictions carried out by genetic modifications as well as RNAi screens, can be rapidly tested. RESULTS: In order to explore the function of a set of C. elegans genes of unknown function, as well as their potential functional roles in the human genome, we performed a phylogenetic analysis to select the most probable worm orthologs. A total of 13 C. elegans genes were subjected to down-regulation via RNAi and characterization of expression profiles using GFP strains. Previously unknown distinct expression patterns were observed for four of the analyzed genes, as well as four visible RNAi phenotypes. In addition, subcellular protein over-expression profiles of the human orthologs for seven out of the thirteen genes using human cells were also analyzed. CONCLUSION: By combining a whole-organism approach using C. elegans with complementary experimental work done on human cell lines, this analysis extends currently available information on the selected set of genes.