In the version of this article initially published online, the EMBL-EBI accession code now given ... more In the version of this article initially published online, the EMBL-EBI accession code now given in the first sentence of the Data Availability section was not yet available. The information has been added in the print, PDF and HTML versions of the article.
Small cell lung cancer (SCLC) is an aggressive malignancy with exceptionally poor prognosis and l... more Small cell lung cancer (SCLC) is an aggressive malignancy with exceptionally poor prognosis and limited therapeutic advances in the past few decades. Although SCLCs are treated as a single disease entity in clinic, emerging data support subtypes of SCLC driven by expression of distinct transcription regulators, which engender unique therapeutic vulnerabilities. However, the translational potential of these observations is limited by access to tumor biopsies. Here, we apply chromatin immunoprecipitation of cell-free nucleosomes carrying active chromatin modifications followed by sequencing (cfChIP-seq) to 299 plasma samples from patients with advanced SCLC, non-SCLC cancers, and healthy adults. We find that cfChIP-seq reliably estimates SCLC circulating free DNA (cfDNA) tumor fraction, recovers the unique epigenetic states of SCLC tissue and cell of origin, and importantly tumor gene expression. Comparison of cfChIP-seq signals to matched tumor transcriptome shows genome-wide concord...
In the version of this article initially published online, the EMBL-EBI accession code now given ... more In the version of this article initially published online, the EMBL-EBI accession code now given in the first sentence of the Data Availability section was not yet available. The information has been added in the print, PDF and HTML versions of the article.
The current SARS-CoV2 epidemic calls for large scale viral tests. The current testing procedure f... more The current SARS-CoV2 epidemic calls for large scale viral tests. The current testing procedure for the presence of virions involves several steps-pharyngeal/nasal swab, cell/viral lysis, RNA extraction, and an rtPCR assay targeting the viral genome and a human gene as an internal sample control. Improvement of any of these steps in terms of cost, processing time, or reagent availability, could result in a significant increase of testing capacity worldwide. Here, we describe a rapid and efficient home made SPRI-based RNA extraction method from a lysed sample. Our approach can be fully automated, is quick (<30 min for 96 samples) and cheap (<1$ per sample), and was tested successfully on more than 250 clinical samples with approved rt-qPCR detection kits. ATTACHMENTS robotic_COVID_SPRI.pdf COVID-19_SPRI_080420.zip
A sequencing-based detection and genotyping assay for SARS-CoV-2 is based on early sample pooling... more A sequencing-based detection and genotyping assay for SARS-CoV-2 is based on early sample pooling using barcoded oligo hybridization.
The global SARS-CoV-2 pandemic created a dire need for viral detection tests worldwide. Most curr... more The global SARS-CoV-2 pandemic created a dire need for viral detection tests worldwide. Most current tests for SARS-CoV-2 are based on RNA extraction followed by quantitative reverse-transcription PCR assays. While automation and improved logistics increased the capacity of these tests, they cannot exceed the lower bound dictated by one extraction and one RT-PCR reaction per sample. Multiplexed next generation sequencing (NGS) assays provide a dramatic increase in throughput, and hold the promise of richer information including viral strains, host immune response, and multiple pathogens.Here, we establish a significant improvement of existing RNA-seq detection protocols. Our workflow, ApharSeq, includes a fast and cheap RNA capture step, that is coupled to barcoding of individual samples, followed by sample-pooling prior to the reverse transcription, PCR and massively parallel sequencing. Thus, only one non-enzymatic step is performed before pooling hundreds of barcoded samples for ...
How transcription factors (TFs) interpret cis-regulatory DNA sequence to control gene expression ... more How transcription factors (TFs) interpret cis-regulatory DNA sequence to control gene expression remains unclear, largely because past studies using native and engineered sequences had insufficient scale. Here, we measure the expression output of >100 million synthetic yeast promoter sequences that are fully random. These sequences yield diverse, reproducible expression levels that can be explained by their chance inclusion of functional TF binding sites. We use machine learning to build interpretable models of transcriptional regulation that predict ~94% of the expression driven from independent test promoters and ~89% of the expression driven from native yeast promoter fragments. These models allow us to characterize each TF's specificity, activity and interactions with chromatin. TF activity depends on binding-site strand, position, DNA helical face and chromatin context. Notably, expression level is influenced by weak regulatory interactions, which confound designed-sequence studies. Our analyses show that massive-throughput assays of fully random DNA can provide the big data necessary to develop complex, predictive models of gene regulation.
Transcription factor (TF) binding to DNA is crucial for transcriptional regulation. There are mul... more Transcription factor (TF) binding to DNA is crucial for transcriptional regulation. There are multiple methods for mapping such binding. These methods balance between input requirements, spatial resolution, and compatibility with high-throughput automation. Here, we describe SLIM-ChIP (short-fragment-enriched, low-input, indexed MNase ChIP), which combines enzymatic fragmentation of chromatin and on-bead indexing to address these desiderata. SLIM-ChIP reproduces a high-resolution binding map of yeast Reb1 comparable with existing methods, yet with less input material and full compatibility with high-throughput procedures. We demonstrate the robustness and flexibility of SLIM-ChIP by probing additional factors in yeast and mouse. Finally, we show that SLIM-ChIP provides information on the chromatin landscape surrounding the bound transcription factor. We identify a class of Reb1 sites where the proximal -1 nucleosome tightly interacts with Reb1 and maintains unidirectional transcript...
Highlights d New reporter-based high-throughput degradation assay was developed d Thousands of de... more Highlights d New reporter-based high-throughput degradation assay was developed d Thousands of degradation signals (degrons) from the yeast transcriptome were identified d A secondary screen revealed multiple targets of the E3 ligase Doa10p d The addition of nuclear localization signal greatly changed degron propensity
Deciphering cis-regulation, the code by which transcription factors (TFs) interpret regulatory DN... more Deciphering cis-regulation, the code by which transcription factors (TFs) interpret regulatory DNA sequence to control gene expression levels, is a long-standing challenge. Previous studies of native or engineered sequences have remained limited in scale. Here, we use random sequences as an alternative, allowing us to measure the expression output of over 100 million synthetic yeast promoters. Random sequences yield a broad range of reproducible expression levels, indicating that the fortuitous binding sites in random DNA are functional. From these data we learn models of transcriptional regulation that predict over 94% of the expression driven from independent test data and nearly 89% from sequences from yeast promoters. These models allow us to characterize the activity of TFs and their interactions with chromatin, and help refine cis-regulatory motifs. We find that strand, position, and helical face preferences of TFs are widespread and depend on interactions with neighboring chr...
Chromatin immunoprecipitation followed by sequencing (ChIP-seq) has been instrumental to our curr... more Chromatin immunoprecipitation followed by sequencing (ChIP-seq) has been instrumental to our current view of chromatin structure and function. It allows genome-wide mapping of histone marks, which demarcate biologically relevant domains. However, ChIP-seq is an ensemble measurement reporting the average occupancy of individual marks in a cell population. Consequently, our understanding of the combinatorial nature of chromatin states relies almost exclusively on correlation between the genomic distributions of individual marks. Here, we report the development of combinatorial-iChIP to determine the genome-wide co-occurrence of histone marks at single-nucleosome resolution. By comparing to a null model, we show that certain combinations of overlapping marks (H3K36me3 and H3K79me3) co-occur more frequently than would be expected by chance, while others (H3K4me3 and H3K36me3) do not, reflecting differences in the underlying chromatin pathways. We further use combinatorial-iChIP to illum...
The liver is the main organ responsible for the modification, clearance, and transformational tox... more The liver is the main organ responsible for the modification, clearance, and transformational toxicity of most xenobiotics due its abundance in CYP450 enzymes. The scarcity and variability of primary hepatocytes currently limits their utility. Human pluripotent stem cells (hPSC) represent an excellent source of differentiated hepatocytes, however, current protocols still produce fetal-like hepatocytes with limited mature function. Interestingly, fetal hepatocytes acquire mature CYP450 expression only post-partum, suggesting that nutritional cues may drive hepatic maturation. We show that vitamin K2 and lithocholic acid, a byproduct of intestinal flora, activate PXR and subsequent CYP3A4 and CYP2C9 expression in hPSC-derived and isolated fetal hepatocytes. Differentiated cells produced albumin and ApoB100 at levels equivalent to primary human hepatocytes, while demonstrating an 8-fold induction of CYP450 activity in response to AhR agonist omeprazole, and a 10-fold induction in response to PXR agonist rifampicin. FACS analysis showed that over 83% of the cells were albumin and HNF4α positive, permitting high-content screening in a 96-well plate format. Analysis of 12 compounds showed an R(2) correlation of 0.94 between TC50 values obtained in stem cell-derived hepatocytes and primary cells, compared to 0.62 for HepG2 cells. Finally, stem cell-derived hepatocytes demonstrate all toxicological end points examined, including steatosis, apoptosis, and cholestasis when exposed to 9 known hepatotoxins. In conclusion, our work provides fresh insights into liver development, suggesting that microbial-derived cues may drive the maturation of CYP450 enzymes post-partum. Addition of these cues resulted in the first functional, inducible, hPSC-derived hepatocyte for predictive toxicology. This article is protected by copyright. All rights reserved.
Two recent studies mapped nucleosomes across the yeast and human genomes, teasing apart the relat... more Two recent studies mapped nucleosomes across the yeast and human genomes, teasing apart the relative contributions of DNA sequence and chromatin remodelers to nucleosome organization. These data suggest two emerging models: chromatin remodelers position nucleosomes around transcriptional start sites in yeast, and a few “locked” nucleosomes may serve as barriers from which nucleosome arrays emanate in human genomes.
Two recent studies mapped nucleosomes across the yeast and human genomes, teasing apart the relat... more Two recent studies mapped nucleosomes across the yeast and human genomes, teasing apart the relative contributions of DNA sequence and chromatin remodelers to nucleosome organization. These data suggest two emerging models: chromatin remodelers position nucleosomes around transcriptional start sites in yeast, while a few 'locked' nucleosomes may serve as barriers from which nucleosome arrays emanate in human genomes.
In the version of this article initially published online, the EMBL-EBI accession code now given ... more In the version of this article initially published online, the EMBL-EBI accession code now given in the first sentence of the Data Availability section was not yet available. The information has been added in the print, PDF and HTML versions of the article.
Small cell lung cancer (SCLC) is an aggressive malignancy with exceptionally poor prognosis and l... more Small cell lung cancer (SCLC) is an aggressive malignancy with exceptionally poor prognosis and limited therapeutic advances in the past few decades. Although SCLCs are treated as a single disease entity in clinic, emerging data support subtypes of SCLC driven by expression of distinct transcription regulators, which engender unique therapeutic vulnerabilities. However, the translational potential of these observations is limited by access to tumor biopsies. Here, we apply chromatin immunoprecipitation of cell-free nucleosomes carrying active chromatin modifications followed by sequencing (cfChIP-seq) to 299 plasma samples from patients with advanced SCLC, non-SCLC cancers, and healthy adults. We find that cfChIP-seq reliably estimates SCLC circulating free DNA (cfDNA) tumor fraction, recovers the unique epigenetic states of SCLC tissue and cell of origin, and importantly tumor gene expression. Comparison of cfChIP-seq signals to matched tumor transcriptome shows genome-wide concord...
In the version of this article initially published online, the EMBL-EBI accession code now given ... more In the version of this article initially published online, the EMBL-EBI accession code now given in the first sentence of the Data Availability section was not yet available. The information has been added in the print, PDF and HTML versions of the article.
The current SARS-CoV2 epidemic calls for large scale viral tests. The current testing procedure f... more The current SARS-CoV2 epidemic calls for large scale viral tests. The current testing procedure for the presence of virions involves several steps-pharyngeal/nasal swab, cell/viral lysis, RNA extraction, and an rtPCR assay targeting the viral genome and a human gene as an internal sample control. Improvement of any of these steps in terms of cost, processing time, or reagent availability, could result in a significant increase of testing capacity worldwide. Here, we describe a rapid and efficient home made SPRI-based RNA extraction method from a lysed sample. Our approach can be fully automated, is quick (<30 min for 96 samples) and cheap (<1$ per sample), and was tested successfully on more than 250 clinical samples with approved rt-qPCR detection kits. ATTACHMENTS robotic_COVID_SPRI.pdf COVID-19_SPRI_080420.zip
A sequencing-based detection and genotyping assay for SARS-CoV-2 is based on early sample pooling... more A sequencing-based detection and genotyping assay for SARS-CoV-2 is based on early sample pooling using barcoded oligo hybridization.
The global SARS-CoV-2 pandemic created a dire need for viral detection tests worldwide. Most curr... more The global SARS-CoV-2 pandemic created a dire need for viral detection tests worldwide. Most current tests for SARS-CoV-2 are based on RNA extraction followed by quantitative reverse-transcription PCR assays. While automation and improved logistics increased the capacity of these tests, they cannot exceed the lower bound dictated by one extraction and one RT-PCR reaction per sample. Multiplexed next generation sequencing (NGS) assays provide a dramatic increase in throughput, and hold the promise of richer information including viral strains, host immune response, and multiple pathogens.Here, we establish a significant improvement of existing RNA-seq detection protocols. Our workflow, ApharSeq, includes a fast and cheap RNA capture step, that is coupled to barcoding of individual samples, followed by sample-pooling prior to the reverse transcription, PCR and massively parallel sequencing. Thus, only one non-enzymatic step is performed before pooling hundreds of barcoded samples for ...
How transcription factors (TFs) interpret cis-regulatory DNA sequence to control gene expression ... more How transcription factors (TFs) interpret cis-regulatory DNA sequence to control gene expression remains unclear, largely because past studies using native and engineered sequences had insufficient scale. Here, we measure the expression output of >100 million synthetic yeast promoter sequences that are fully random. These sequences yield diverse, reproducible expression levels that can be explained by their chance inclusion of functional TF binding sites. We use machine learning to build interpretable models of transcriptional regulation that predict ~94% of the expression driven from independent test promoters and ~89% of the expression driven from native yeast promoter fragments. These models allow us to characterize each TF's specificity, activity and interactions with chromatin. TF activity depends on binding-site strand, position, DNA helical face and chromatin context. Notably, expression level is influenced by weak regulatory interactions, which confound designed-sequence studies. Our analyses show that massive-throughput assays of fully random DNA can provide the big data necessary to develop complex, predictive models of gene regulation.
Transcription factor (TF) binding to DNA is crucial for transcriptional regulation. There are mul... more Transcription factor (TF) binding to DNA is crucial for transcriptional regulation. There are multiple methods for mapping such binding. These methods balance between input requirements, spatial resolution, and compatibility with high-throughput automation. Here, we describe SLIM-ChIP (short-fragment-enriched, low-input, indexed MNase ChIP), which combines enzymatic fragmentation of chromatin and on-bead indexing to address these desiderata. SLIM-ChIP reproduces a high-resolution binding map of yeast Reb1 comparable with existing methods, yet with less input material and full compatibility with high-throughput procedures. We demonstrate the robustness and flexibility of SLIM-ChIP by probing additional factors in yeast and mouse. Finally, we show that SLIM-ChIP provides information on the chromatin landscape surrounding the bound transcription factor. We identify a class of Reb1 sites where the proximal -1 nucleosome tightly interacts with Reb1 and maintains unidirectional transcript...
Highlights d New reporter-based high-throughput degradation assay was developed d Thousands of de... more Highlights d New reporter-based high-throughput degradation assay was developed d Thousands of degradation signals (degrons) from the yeast transcriptome were identified d A secondary screen revealed multiple targets of the E3 ligase Doa10p d The addition of nuclear localization signal greatly changed degron propensity
Deciphering cis-regulation, the code by which transcription factors (TFs) interpret regulatory DN... more Deciphering cis-regulation, the code by which transcription factors (TFs) interpret regulatory DNA sequence to control gene expression levels, is a long-standing challenge. Previous studies of native or engineered sequences have remained limited in scale. Here, we use random sequences as an alternative, allowing us to measure the expression output of over 100 million synthetic yeast promoters. Random sequences yield a broad range of reproducible expression levels, indicating that the fortuitous binding sites in random DNA are functional. From these data we learn models of transcriptional regulation that predict over 94% of the expression driven from independent test data and nearly 89% from sequences from yeast promoters. These models allow us to characterize the activity of TFs and their interactions with chromatin, and help refine cis-regulatory motifs. We find that strand, position, and helical face preferences of TFs are widespread and depend on interactions with neighboring chr...
Chromatin immunoprecipitation followed by sequencing (ChIP-seq) has been instrumental to our curr... more Chromatin immunoprecipitation followed by sequencing (ChIP-seq) has been instrumental to our current view of chromatin structure and function. It allows genome-wide mapping of histone marks, which demarcate biologically relevant domains. However, ChIP-seq is an ensemble measurement reporting the average occupancy of individual marks in a cell population. Consequently, our understanding of the combinatorial nature of chromatin states relies almost exclusively on correlation between the genomic distributions of individual marks. Here, we report the development of combinatorial-iChIP to determine the genome-wide co-occurrence of histone marks at single-nucleosome resolution. By comparing to a null model, we show that certain combinations of overlapping marks (H3K36me3 and H3K79me3) co-occur more frequently than would be expected by chance, while others (H3K4me3 and H3K36me3) do not, reflecting differences in the underlying chromatin pathways. We further use combinatorial-iChIP to illum...
The liver is the main organ responsible for the modification, clearance, and transformational tox... more The liver is the main organ responsible for the modification, clearance, and transformational toxicity of most xenobiotics due its abundance in CYP450 enzymes. The scarcity and variability of primary hepatocytes currently limits their utility. Human pluripotent stem cells (hPSC) represent an excellent source of differentiated hepatocytes, however, current protocols still produce fetal-like hepatocytes with limited mature function. Interestingly, fetal hepatocytes acquire mature CYP450 expression only post-partum, suggesting that nutritional cues may drive hepatic maturation. We show that vitamin K2 and lithocholic acid, a byproduct of intestinal flora, activate PXR and subsequent CYP3A4 and CYP2C9 expression in hPSC-derived and isolated fetal hepatocytes. Differentiated cells produced albumin and ApoB100 at levels equivalent to primary human hepatocytes, while demonstrating an 8-fold induction of CYP450 activity in response to AhR agonist omeprazole, and a 10-fold induction in response to PXR agonist rifampicin. FACS analysis showed that over 83% of the cells were albumin and HNF4α positive, permitting high-content screening in a 96-well plate format. Analysis of 12 compounds showed an R(2) correlation of 0.94 between TC50 values obtained in stem cell-derived hepatocytes and primary cells, compared to 0.62 for HepG2 cells. Finally, stem cell-derived hepatocytes demonstrate all toxicological end points examined, including steatosis, apoptosis, and cholestasis when exposed to 9 known hepatotoxins. In conclusion, our work provides fresh insights into liver development, suggesting that microbial-derived cues may drive the maturation of CYP450 enzymes post-partum. Addition of these cues resulted in the first functional, inducible, hPSC-derived hepatocyte for predictive toxicology. This article is protected by copyright. All rights reserved.
Two recent studies mapped nucleosomes across the yeast and human genomes, teasing apart the relat... more Two recent studies mapped nucleosomes across the yeast and human genomes, teasing apart the relative contributions of DNA sequence and chromatin remodelers to nucleosome organization. These data suggest two emerging models: chromatin remodelers position nucleosomes around transcriptional start sites in yeast, and a few “locked” nucleosomes may serve as barriers from which nucleosome arrays emanate in human genomes.
Two recent studies mapped nucleosomes across the yeast and human genomes, teasing apart the relat... more Two recent studies mapped nucleosomes across the yeast and human genomes, teasing apart the relative contributions of DNA sequence and chromatin remodelers to nucleosome organization. These data suggest two emerging models: chromatin remodelers position nucleosomes around transcriptional start sites in yeast, while a few 'locked' nucleosomes may serve as barriers from which nucleosome arrays emanate in human genomes.
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