The mammary epithelial cell (MEC) system is a bilayered ductal epithelium of luminal and basal ce... more The mammary epithelial cell (MEC) system is a bilayered ductal epithelium of luminal and basal cells, maintained by a lineage of stem and progenitor populations. Here, we used integrated singlecell transcriptomics and chromatin accessibility analysis to reconstruct the cell types of the mouse MEC system and their underlying gene regulatory features in an unbiased manner. We define differentiation states within the secretory type of luminal cells, which forms a continuous spectrum of general luminal progenitor and lactation-committed progenitor cells. By integrating single-cell transcriptomics and chromatin accessibility landscapes, we identify cisand trans-regulatory elements that are differentially activated in the specific epithelial cell types and our newly defined luminal differentiation states. Our work provides a resource to reveal cis/trans-regulatory elements associated with MEC identity and differentiation that will serve as a reference to determine how the chromatin accessibility landscape changes during breast cancer.
Thank you again for submitting your work to Molecular Systems Biology. We have now heard back fro... more Thank you again for submitting your work to Molecular Systems Biology. We have now heard back from the three referees who agreed to evaluate your manuscript. Overall, the reviewers acknowledge that the presented analysis is comprehensive and results in potentially interesting findings. However, as you will see from the reports below, they raise concerns on your work, which should be convincingly addressed in a revision of the manuscript.
The comparison of gene regulatory networks between diseased versus healthy individuals or between... more The comparison of gene regulatory networks between diseased versus healthy individuals or between two different treatments is an important scientific problem. Here, we propose sc-compReg as a method for the comparative analysis of gene expression regulatory networks between two conditions using single cell gene expression (scRNA-seq) and single cell chromatin accessibility data (scATAC-seq). Our software, sc-compReg, can be used as a stand-alone package that provides joint clustering and embedding of the cells from both scRNA-seq and scATAC-seq, and the construction of differential regulatory networks across two conditions. We apply the method to compare the gene regulatory networks of an individual with chronic lymphocytic leukemia (CLL) versus a healthy control. The analysis reveals a tumor-specific B cell subpopulation in the CLL patient and identifies TOX2 as a potential regulator of this subpopulation.
ABSTRACTThe mammary epithelial cell (MEC) system is a bi-layered ductal epithelial network consis... more ABSTRACTThe mammary epithelial cell (MEC) system is a bi-layered ductal epithelial network consisting of luminal and basal cells, which is maintained by a lineage of stem and progenitor cell populations. Here, we used integrated single-cell transcriptomics and chromatin accessibility analysis to reconstruct the cell types of the mouse MEC system and their underlying gene regulatory features in an unbiased manner. We define previously unrealized differentiation states within the secretory type of luminal cells, which can be divided into distinct clusters of progenitor and mature secretory cells. By integrating single-cell transcriptomics and chromatin accessibility landscapes, we identified novel cis- and trans-regulatory elements that are differentially activated in the specific epithelial cell types and our newly defined luminal differentiation states. Our work provides an unprecedented resource to reveal novel cis/trans regulatory elements associated with MEC identity and differen...
Understanding complex tissues requires single-cell deconstruction of gene regulation with precisi... more Understanding complex tissues requires single-cell deconstruction of gene regulation with precision and scale. Here we present a massively parallel droplet-based platform for mapping transposase-accessible chromatin in tens of thousands of single cells per sample (scATAC-seq). We obtain and analyze chromatin profiles of over 200,000 single cells in two primary human systems. In blood, scATAC-seq allows marker-free identification of cell type-specificcis- andtrans-regulatory elements, mapping of disease-associated enhancer activity, and reconstruction of trajectories of differentiation from progenitors to diverse and rare immune cell types. In basal cell carcinoma, scATAC-seq reveals regulatory landscapes of malignant, stromal, and immune cell types in the tumor microenvironment. Moreover, scATAC-seq of serial tumor biopsies before and after PD-1 blockade allows identification of chromatin regulators and differentiation trajectories of therapy-responsive intratumoral T cell subsets, ...
Proceedings of the National Academy of Sciences of the United States of America, May 2, 2017
The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) orchestra... more The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) orchestrates dynamic recruitment of specific cellular machines during different stages of transcription. Signature phosphorylation patterns of Y1S2P3T4S5P6S7 heptapeptide repeats of the CTD engage specific "readers." Whereas phospho-Ser5 and phospho-Ser2 marks are ubiquitous, phospho-Thr4 is reported to only impact specific genes. Here, we identify a role for phospho-Thr4 in transcription termination at noncoding small nucleolar RNA (snoRNA) genes. Quantitative proteomics reveals an interactome of known readers as well as protein complexes that were not known to rely on Thr4 for association with Pol II. The data indicate a key role for Thr4 in engaging the machinery used for transcription elongation and termination. We focus on Rtt103, a protein that binds phospho-Ser2 and phospho-Thr4 marks and facilitates transcription termination at protein-coding genes. To elucidate how Rtt103 engag...
The C-terminal domain (CTD) of RNA polymerase II (Pol II) consists of conserved heptapeptide repe... more The C-terminal domain (CTD) of RNA polymerase II (Pol II) consists of conserved heptapeptide repeats that function as a binding platform for different protein complexes involved in transcription, RNA processing, export, and chromatin remodeling. The CTD repeats are subject to sequential waves of posttranslational modifications during specific stages of the transcription cycle. These patterned modifications have led to the postulation of the “CTD code” hypothesis, where stage-specific patterns define a spatiotemporal code that is recognized by the appropriate interacting partners. Here, we highlight the role of CTD modifications in directing transcription initiation, elongation, and termination. We examine the major readers, writers, and erasers of the CTD code and examine the relevance of describing patterns of posttranslational modifications as a “code.” Finally, we discuss major questions regarding the function of the newly discovered CTD modifications and the fundamental insights...
Stressed cells coordinate a multi-faceted response spanning many levels of physiology. Yet knowle... more Stressed cells coordinate a multi-faceted response spanning many levels of physiology. Yet knowledge of the complete stress-activated regulatory network as well as design principles for signal integration remains incomplete. We developed an experimental and computational approach to integrate available protein interaction data with gene fitness contributions, mutant transcriptome profiles, and phospho-proteome changes in cells responding to salt stress, to infer the salt-responsive signaling network in yeast. The inferred subnetwork presented many novel predictions by implicating new regulators, uncovering unrecognized crosstalk between known pathways, and pointing to previously unknown 'hubs' of signal integration. We exploited these predictions to show that Cdc14 phosphatase is a central hub in the network and that modification of RNA polymerase II coordinates induction of stress-defense genes with reduction of growth-related transcripts. We find that the orthologous human...
Phosphorylation of the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II... more Phosphorylation of the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) governs stage-specific interactions with different cellular machines. The CTD consists of YSPTSPS heptad repeats and sequential phosphorylations of Ser7, Ser5 and Ser2 occur universally at Pol II-transcribed genes. Phosphorylation of Thr4, however, appears to selectively modulate transcription of specific classes of genes. Here, we identify ten new Thr4 kinases from different kinase structural groups. Irreversible chemical inhibition of the most active Thr4 kinase, Hrr25, reveals a novel role for this kinase in transcription termination of specific class of noncoding snoRNA genes. Genome-wide profiles of Hrr25 reveal a selective enrichment at 3' regions of noncoding genes that display termination defects. Importantly, phospho-Thr4 marks placed by Hrr25 are recognized by Rtt103, a key component of the termination machinery. Our results suggest that these uncommon CTD kinases...
Stressed cells coordinate a multi-faceted response spanning many levels of physiology. Yet knowle... more Stressed cells coordinate a multi-faceted response spanning many levels of physiology. Yet knowledge of the complete stress-activated regulatory network as well as design principles for signal integration remains incomplete. We developed an experimental and computational approach to integrate available protein interaction data with gene fitness contributions, mutant transcriptome profiles, and phospho-proteome changes in cells responding to salt stress, to infer the salt-responsive signaling network in yeast. The inferred subnetwork presented many novel predictions by implicating new regulators, uncovering unrecognized crosstalk between known pathways, and pointing to previously unknown 'hubs' of signal integration. We exploited these predictions to show that Cdc14 phosphatase is a central hub in the network and that modification of RNA polymerase II coordinates induction of stress-defense genes with reduction of growth-related transcripts. We find that the orthologous human...
The mammary epithelial cell (MEC) system is a bilayered ductal epithelium of luminal and basal ce... more The mammary epithelial cell (MEC) system is a bilayered ductal epithelium of luminal and basal cells, maintained by a lineage of stem and progenitor populations. Here, we used integrated singlecell transcriptomics and chromatin accessibility analysis to reconstruct the cell types of the mouse MEC system and their underlying gene regulatory features in an unbiased manner. We define differentiation states within the secretory type of luminal cells, which forms a continuous spectrum of general luminal progenitor and lactation-committed progenitor cells. By integrating single-cell transcriptomics and chromatin accessibility landscapes, we identify cisand trans-regulatory elements that are differentially activated in the specific epithelial cell types and our newly defined luminal differentiation states. Our work provides a resource to reveal cis/trans-regulatory elements associated with MEC identity and differentiation that will serve as a reference to determine how the chromatin accessibility landscape changes during breast cancer.
Thank you again for submitting your work to Molecular Systems Biology. We have now heard back fro... more Thank you again for submitting your work to Molecular Systems Biology. We have now heard back from the three referees who agreed to evaluate your manuscript. Overall, the reviewers acknowledge that the presented analysis is comprehensive and results in potentially interesting findings. However, as you will see from the reports below, they raise concerns on your work, which should be convincingly addressed in a revision of the manuscript.
The comparison of gene regulatory networks between diseased versus healthy individuals or between... more The comparison of gene regulatory networks between diseased versus healthy individuals or between two different treatments is an important scientific problem. Here, we propose sc-compReg as a method for the comparative analysis of gene expression regulatory networks between two conditions using single cell gene expression (scRNA-seq) and single cell chromatin accessibility data (scATAC-seq). Our software, sc-compReg, can be used as a stand-alone package that provides joint clustering and embedding of the cells from both scRNA-seq and scATAC-seq, and the construction of differential regulatory networks across two conditions. We apply the method to compare the gene regulatory networks of an individual with chronic lymphocytic leukemia (CLL) versus a healthy control. The analysis reveals a tumor-specific B cell subpopulation in the CLL patient and identifies TOX2 as a potential regulator of this subpopulation.
ABSTRACTThe mammary epithelial cell (MEC) system is a bi-layered ductal epithelial network consis... more ABSTRACTThe mammary epithelial cell (MEC) system is a bi-layered ductal epithelial network consisting of luminal and basal cells, which is maintained by a lineage of stem and progenitor cell populations. Here, we used integrated single-cell transcriptomics and chromatin accessibility analysis to reconstruct the cell types of the mouse MEC system and their underlying gene regulatory features in an unbiased manner. We define previously unrealized differentiation states within the secretory type of luminal cells, which can be divided into distinct clusters of progenitor and mature secretory cells. By integrating single-cell transcriptomics and chromatin accessibility landscapes, we identified novel cis- and trans-regulatory elements that are differentially activated in the specific epithelial cell types and our newly defined luminal differentiation states. Our work provides an unprecedented resource to reveal novel cis/trans regulatory elements associated with MEC identity and differen...
Understanding complex tissues requires single-cell deconstruction of gene regulation with precisi... more Understanding complex tissues requires single-cell deconstruction of gene regulation with precision and scale. Here we present a massively parallel droplet-based platform for mapping transposase-accessible chromatin in tens of thousands of single cells per sample (scATAC-seq). We obtain and analyze chromatin profiles of over 200,000 single cells in two primary human systems. In blood, scATAC-seq allows marker-free identification of cell type-specificcis- andtrans-regulatory elements, mapping of disease-associated enhancer activity, and reconstruction of trajectories of differentiation from progenitors to diverse and rare immune cell types. In basal cell carcinoma, scATAC-seq reveals regulatory landscapes of malignant, stromal, and immune cell types in the tumor microenvironment. Moreover, scATAC-seq of serial tumor biopsies before and after PD-1 blockade allows identification of chromatin regulators and differentiation trajectories of therapy-responsive intratumoral T cell subsets, ...
Proceedings of the National Academy of Sciences of the United States of America, May 2, 2017
The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) orchestra... more The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) orchestrates dynamic recruitment of specific cellular machines during different stages of transcription. Signature phosphorylation patterns of Y1S2P3T4S5P6S7 heptapeptide repeats of the CTD engage specific "readers." Whereas phospho-Ser5 and phospho-Ser2 marks are ubiquitous, phospho-Thr4 is reported to only impact specific genes. Here, we identify a role for phospho-Thr4 in transcription termination at noncoding small nucleolar RNA (snoRNA) genes. Quantitative proteomics reveals an interactome of known readers as well as protein complexes that were not known to rely on Thr4 for association with Pol II. The data indicate a key role for Thr4 in engaging the machinery used for transcription elongation and termination. We focus on Rtt103, a protein that binds phospho-Ser2 and phospho-Thr4 marks and facilitates transcription termination at protein-coding genes. To elucidate how Rtt103 engag...
The C-terminal domain (CTD) of RNA polymerase II (Pol II) consists of conserved heptapeptide repe... more The C-terminal domain (CTD) of RNA polymerase II (Pol II) consists of conserved heptapeptide repeats that function as a binding platform for different protein complexes involved in transcription, RNA processing, export, and chromatin remodeling. The CTD repeats are subject to sequential waves of posttranslational modifications during specific stages of the transcription cycle. These patterned modifications have led to the postulation of the “CTD code” hypothesis, where stage-specific patterns define a spatiotemporal code that is recognized by the appropriate interacting partners. Here, we highlight the role of CTD modifications in directing transcription initiation, elongation, and termination. We examine the major readers, writers, and erasers of the CTD code and examine the relevance of describing patterns of posttranslational modifications as a “code.” Finally, we discuss major questions regarding the function of the newly discovered CTD modifications and the fundamental insights...
Stressed cells coordinate a multi-faceted response spanning many levels of physiology. Yet knowle... more Stressed cells coordinate a multi-faceted response spanning many levels of physiology. Yet knowledge of the complete stress-activated regulatory network as well as design principles for signal integration remains incomplete. We developed an experimental and computational approach to integrate available protein interaction data with gene fitness contributions, mutant transcriptome profiles, and phospho-proteome changes in cells responding to salt stress, to infer the salt-responsive signaling network in yeast. The inferred subnetwork presented many novel predictions by implicating new regulators, uncovering unrecognized crosstalk between known pathways, and pointing to previously unknown 'hubs' of signal integration. We exploited these predictions to show that Cdc14 phosphatase is a central hub in the network and that modification of RNA polymerase II coordinates induction of stress-defense genes with reduction of growth-related transcripts. We find that the orthologous human...
Phosphorylation of the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II... more Phosphorylation of the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) governs stage-specific interactions with different cellular machines. The CTD consists of YSPTSPS heptad repeats and sequential phosphorylations of Ser7, Ser5 and Ser2 occur universally at Pol II-transcribed genes. Phosphorylation of Thr4, however, appears to selectively modulate transcription of specific classes of genes. Here, we identify ten new Thr4 kinases from different kinase structural groups. Irreversible chemical inhibition of the most active Thr4 kinase, Hrr25, reveals a novel role for this kinase in transcription termination of specific class of noncoding snoRNA genes. Genome-wide profiles of Hrr25 reveal a selective enrichment at 3' regions of noncoding genes that display termination defects. Importantly, phospho-Thr4 marks placed by Hrr25 are recognized by Rtt103, a key component of the termination machinery. Our results suggest that these uncommon CTD kinases...
Stressed cells coordinate a multi-faceted response spanning many levels of physiology. Yet knowle... more Stressed cells coordinate a multi-faceted response spanning many levels of physiology. Yet knowledge of the complete stress-activated regulatory network as well as design principles for signal integration remains incomplete. We developed an experimental and computational approach to integrate available protein interaction data with gene fitness contributions, mutant transcriptome profiles, and phospho-proteome changes in cells responding to salt stress, to infer the salt-responsive signaling network in yeast. The inferred subnetwork presented many novel predictions by implicating new regulators, uncovering unrecognized crosstalk between known pathways, and pointing to previously unknown 'hubs' of signal integration. We exploited these predictions to show that Cdc14 phosphatase is a central hub in the network and that modification of RNA polymerase II coordinates induction of stress-defense genes with reduction of growth-related transcripts. We find that the orthologous human...
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