Genome-wide Transcription Factor Binding Research Articles

Quantifying genome-wide transcription factor binding affinities for chromatin using BANC-seq.

Transcription factors (TFs) bind specific DNA sequences to regulate transcription. Apart from DNA sequences, local factors such as DNA accessibility and chromatin structure determine the affinity of a TF for any given locus. Including these factors when measuring TF-DNA affinities has proven difficult. To address this challenge, we recently developed a method called binding affinities in native chromatin by sequencing (BANC-seq). In BANC-seq, intact mammalian nuclei are incubated with a concentration range of epitope-tagged TF, followed by either chromatin immunoprecipitation or cleavage under target and release using nuclease with spike-in DNA. This allows determination of apparent dissociation constant (KdApp) values, defined by the concentration of TF at which half-maximum binding occurs, across the genome. Here we present a detailed stepwise protocol for BANC-seq, including downstream data analysis. In principle, any molecular biologist should be able to perform a BANC-seq experiment in as little as 1.5 d (excluding analysis). However, preprocessing and analysis of the sequencing data does require some experience in command-line shell and R programming.

HNF4A guides the MLL4 complex to establish and maintain H3K4me1 at gene regulatory elements

Hepatocyte nuclear factor 4A (HNF4A/NR2a1), a transcriptional regulator of hepatocyte identity, controls genes that are crucial for liver functions, primarily through binding to enhancers. In mammalian cells, active and primed enhancers are marked by monomethylation of histone 3 (H3) at lysine 4 (K4) (H3K4me1) in a cell type-specific manner. How this modification is established and maintained at enhancers in connection with transcription factors (TFs) remains unknown. Using analysis of genome-wide histone modifications, TF binding, chromatin accessibility and gene expression, we show that HNF4A is essential for an active chromatin state. Using HNF4A loss and gain of function experiments in vivo and in cell lines in vitro, we show that HNF4A affects H3K4me1, H3K27ac and chromatin accessibility, highlighting its contribution to the establishment and maintenance of a transcriptionally permissive epigenetic state. Mechanistically, HNF4A interacts with the mixed-lineage leukaemia 4 (MLL4) complex facilitating recruitment to HNF4A-bound regions. Our findings indicate that HNF4A enriches H3K4me1, H3K27ac and establishes chromatin opening at transcriptional regulatory regions.

Mapping enhancer and chromatin accessibility landscapes charts the regulatory network of Alzheimer's disease

BackgroundEnhancers are regulatory elements that target and modulate gene expression and play a role in human health and disease. However, the roles of enhancer regulatory circuit abnormalities driven by epigenetic alterations in Alzheimer's disease (AD) are unclear. MethodsIn this study, a multiomic integrative analysis was performed to map enhancer and chromatin accessibility landscapes and identify regulatory network abnormalities in AD. We identified differentially methylated enhancers and constructed regulatory networks across brain regions using AD brain tissue samples. Through the integration of snATAC-seq and snRNA-seq datasets, we mapped enhancers with DNA methylation alterations (DMA) and chromatin accessibility landscapes. Core regulatory triplets that contributed to AD neuropathology in specific cell types were further prioritized. ResultsWe revealed widespread DNA methylation alterations (DMA) in the enhancers of AD patients across different brain regions. In addition, the genome-wide transcription factor (TF) binding profiles showed that enhancers with DMA are pervasively regulated by TFs. The TF-enhancer-gene regulatory network analysis identified core regulatory triplets that are associated with brain and immune cell proportions and play important roles in AD pathogenesis. Enhancer regulatory circuits with DMA exhibited distinct chromatin accessibility patterns, which were further characterized at single-cell resolutions. ConclusionsOur study comprehensively investigated DNA methylation-mediated regulatory circuit abnormalities and provided novel insights into the potential pathogenesis of AD.

BloodChIP Xtra: an expanded database of comparative genome-wide transcription factor binding and gene-expression profiles in healthy human stem/progenitor subsets and leukemic cells.

The BloodChIP Xtra database (http://bloodchipXtra.vafaeelab.com/) facilitates genome-wide exploration and visualization of transcription factor (TF) occupancy and chromatin configuration in rare primary human hematopoietic stem (HSC-MPP) and progenitor (CMP, GMP, MEP) cells and acute myeloid leukemia (AML) cell lines (KG-1, ME-1, Kasumi1, TSU-1621-MT), along with chromatin accessibility and gene expression data from these and primary patient AMLs. BloodChIP Xtra features significantly more datasets than our earlier database BloodChIP (two primary cell types and two cell lines). Improved methodologies for determining TF occupancy and chromatin accessibility have led to increased availability of data for rare primary cell types across the spectrum of healthy and AML hematopoiesis. However, there is a continuing need for these data to be integrated in an easily accessible manner for gene-based queries and use in downstream applications. Here, we provide a user-friendly database based around genome-wide binding profiles of key hematopoietic TFs and histone marks in healthy stem/progenitor cell types. These are compared with binding profiles and chromatin accessibility derived from primary and cell line AML and integrated with expression data from corresponding cell types. All queries can be exported to construct TF-gene and protein-protein networks and evaluate the association of genes with specific cellular processes.

Functional investigation of five R2R3-MYB transcription factors associated with wood development in Eucalyptus using DAP-seq-ML.

A multi-tiered transcriptional network regulates xylem differentiation and secondary cell wall (SCW) formation in plants, with evidence of both conserved and lineage-specific SCW network architecture. We aimed to elucidate the roles of selected R2R3-MYB transcription factors (TFs) linked to Eucalyptus wood formation by identifying genome-wide TF binding sites and direct target genes through an improved DAP-seq protocol combined with machine learning for target gene assignment (DAP-seq-ML). We applied this to five TFs including a well-studied SCW master regulator (EgrMYB2; homolog of AtMYB83), a repressor of lignification (EgrMYB1; homolog of AtMYB4), a TF affecting SCW thickness and vessel density (EgrMYB137; homolog of PtrMYB074) and two TFs with unclear roles in SCW regulation (EgrMYB135 and EgrMYB122). Each DAP-seq TF peak set (average 12,613 peaks) was enriched for canonical R2R3-MYB binding motifs. To improve the reliability of target gene assignment to peaks, a random forest classifier was developed from Arabidopsis DAP-seq, RNA-seq, chromatin, and conserved noncoding sequence data which demonstrated significantly higher precision and recall to the baseline method of assigning genes to proximal peaks. EgrMYB1, EgrMYB2 and EgrMYB137 predicted targets showed clear enrichment for SCW-related biological processes. As validation, EgrMYB137 overexpression in transgenic Eucalyptus hairy roots increased xylem lignification, while its dominant repression in transgenic Arabidopsis and Populus reduced xylem lignification, stunted growth, and caused downregulation of SCW genes. EgrMYB137 targets overlapped significantly with those of EgrMYB2, suggesting partial functional redundancy. Our results show that DAP-seq-ML identified biologically relevant R2R3-MYB targets supported by the finding that EgrMYB137 promotes SCW lignification in planta.

Genome-wide transcription factor–binding maps reveal cell-specific changes in the regulatory architecture of human HSPCs

AbstractHematopoietic stem and progenitor cells (HSPCs) rely on a complex interplay among transcription factors (TFs) to regulate differentiation into mature blood cells. A heptad of TFs (FLI1, ERG, GATA2, RUNX1, TAL1, LYL1, LMO2) bind regulatory elements in bulk CD34+ HSPCs. However, whether specific heptad-TF combinations have distinct roles in regulating hematopoietic differentiation remains unknown. We mapped genome-wide chromatin contacts (HiC, H3K27ac, HiChIP), chromatin modifications (H3K4me3, H3K27ac, H3K27me3) and 10 TF binding profiles (heptad, PU.1, CTCF, STAG2) in HSPC subsets (stem/multipotent progenitors plus common myeloid, granulocyte macrophage, and megakaryocyte erythrocyte progenitors) and found TF occupancy and enhancer-promoter interactions varied significantly across cell types and were associated with cell-type–specific gene expression. Distinct regulatory elements were enriched with specific heptad-TF combinations, including stem-cell–specific elements with ERG, and myeloid- and erythroid-specific elements with combinations of FLI1, RUNX1, GATA2, TAL1, LYL1, and LMO2. Furthermore, heptad-occupied regions in HSPCs were subsequently bound by lineage-defining TFs, including PU.1 and GATA1, suggesting that heptad factors may prime regulatory elements for use in mature cell types. We also found that enhancers with cell-type–specific heptad occupancy shared a common grammar with respect to TF binding motifs, suggesting that combinatorial binding of TF complexes was at least partially regulated by features encoded in DNA sequence motifs. Taken together, this study comprehensively characterizes the gene regulatory landscape in rare subpopulations of human HSPCs. The accompanying data sets should serve as a valuable resource for understanding adult hematopoiesis and a framework for analyzing aberrant regulatory networks in leukemic cells.

An Efficient Protocol for CUT&RUN Analysis of FACS-Isolated Mouse Satellite Cells.

Genome-wide analyses with small cell populations are a major constraint for studies, particularly in the stem cell field. This work describes an efficient protocol for the fluorescence-activated cell sorting (FACS) isolation of satellite cells from the limb muscle, a tissue with a high content of structural proteins. Dissected limb muscles from adult mice were mechanically disrupted by mincing in medium supplemented with dispase and type I collagenase. Upon digestion,the homogenate was filtered through cell strainers, and cells were suspended in FACS buffer. Viability was determined with fixable viability stain, and immunostained satellite cells were isolated by FACS. Cells were lysed with Triton X-100 and released nuclei were bound to concanavalin A magnetic beads. Nucleus/bead complexes were incubated with antibodies against the transcription factor or histone modifications of interest. After washes, nucleus/bead complexes were incubated with protein A-micrococcal nuclease, and chromatin cleavage was initiated with CaCl2. After DNA extraction, libraries were generated and sequenced, and the profiles for genome-wide transcription factor binding and covalent histone modifications were obtained by bioinformatic analysis. The peaks obtained for the various histone marks showed that the binding events were specific for satellite cells. Moreover, known motif analysis unveiled that the transcription factor was bound to chromatin via its cognate response element. This protocol is therefore adapted to study gene regulation in adult mice limb muscle satellite cells.

Tet2 coordinates with Foxo1 and Runx1 to balance T follicular helper cell and T helper 1 cell differentiation.

In response to various types of infection, naïve CD4+ T cells differentiate into diverse helper T cell subsets; however, the epigenetic programs that regulate differentiation in response to viral infection remain poorly understood. Demethylation of CpG dinucleotides by Tet methylcytosine dioxygenases is a key component of epigenetic programing that promotes specific gene expression, cellular differentiation, and function. We report that following viral infection, Tet2-deficient CD4+ T cells preferentially differentiate into highly functional germinal center T follicular helper (TFH) cells that provide enhanced help for B cells. Using genome-wide DNA methylation and transcription factor binding analyses, we find that Tet2 coordinates with multiple transcription factors, including Foxo1 and Runx1, to mediate the demethylation and expression of target genes, including genes encoding repressors of TFH differentiation. Our findings establish Tet2 as an important regulator of TFH cell differentiation and reveal pathways that could be targeted to enhance immune responses against infectious disease.

Tet2-mediated programing balances T follicular helper cell and T helper 1 cell differentiation.

Abstract The differentiation of CD4+ T cell subsets in response to infection has been studied extensively, however the epigenetic programs that regulate these processes remain poorly understood. Active demethylation by tet methylcytosine dioxygenases of CpG dinucleotides within DNA is a key component of epigenetic programing that promotes lineage specific gene expression and contributes to cellular differentiation and function. Here we report that Tet2 acts to restrict the differentiation of T follicular helper (Tfh) cells in CD4+ T cells responding to viral infection. Using an adoptive transfer model of virus-specific CD4+ cells we found that Tet2-deficient CD4+ T cells skew away from the Th1 lineage and instead preferentially differentiate into highly functional germinal center (GC) Tfh cells that provide enhanced help for B cell responses. We found that the impact of Tet2-mediated programing on CD4+ T cell differentiation is cell intrinsic and the shift in lineage differentiation occurs as early as 2 days post infection. Using genome-wide expression, DNA methylation and transcription factor binding analyses, we found that Tet2 coordinates with multiple transcription factors to mediate the demethylation and expression of their target genes following activation. Our findings establish Tet2 as an important regulator of Tfh cell differentiation and reveal pathways that could be targeted to enhance GC responses against infectious disease. Supported by grants from NIH (R01 AI137238 to J.S.H., T32 AI055434 to L.M.S., and T32 AI138945 to A.B.

Genome-wide Profiling of Transcription Factor-DNA Binding Interactions in Candida albicans: A Comprehensive CUT&RUN Method and Data Analysis Workflow.

Regulatory transcription factors control many important biological processes, including cellular differentiation, responses to environmental perturbations and stresses, and host-pathogen interactions. Determining the genome-wide binding of regulatory transcription factors to DNA is essential to understanding the function of transcription factors in these often complex biological processes. Cleavage under targets and release using nuclease (CUT&RUN) is a modern method for genome-wide mapping of in vivo protein-DNA binding interactions that is an attractive alternative to the traditional and widely used chromatin immunoprecipitation followed by sequencing (ChIP-seq) method. CUT&RUN is amenable to a higher-throughput experimental setup and has a substantially higher dynamic range with lower per-sample sequencing costs than ChIP-seq. Here, a comprehensive CUT&RUN protocol and accompanying data analysis workflow tailored for genome-wide analysis of transcription factor-DNA binding interactions in the human fungal pathogen Candida albicans are described. This detailed protocol includes all necessary experimental procedures, from epitope tagging of transcription factor-coding genes to library preparation for sequencing; additionally, it includes a customized computational workflow for CUT&RUN data analysis.

Factorbook: an updated catalog of transcription factor motifs and candidate regulatory motif sites.

The human genome contains ∼2000 transcriptional regulatory proteins, including ∼1600 DNA-binding transcription factors (TFs) recognizing characteristic sequence motifs to exert regulatory effects on gene expression. The binding specificities of these factors have been profiled both in vitro, using techniques such as HT-SELEX, and in vivo, using techniques including ChIP-seq. We previously developed Factorbook, a TF-centric database of annotations, motifs, and integrative analyses based on ChIP-seq data from Phase II of the ENCODE Project. Here we present an update to Factorbook which significantly expands the breadth of cell type and TF coverage. The update includes an expanded motif catalog derived from thousands of ENCODE Phase II and III ChIP-seq experiments and HT-SELEX experiments; this motif catalog is integrated with the ENCODE registry of candidate cis-regulatory elements to annotate a comprehensive collection of genome-wide candidate TF binding sites. The database also offers novel tools for applying the motif models within machine learning frameworks and using these models for integrative analysis, including annotation of variants and disease and trait heritability. Factorbook is publicly available at www.factorbook.org; we will continue to expand the resource as ENCODE Phase IV data are released.

Abstract 2189: Subtype-specific epigenomic landscape and 3D genome structure in bladder cancer

Abstract Urinary bladder cancers (BLCA) are commonly diagnosed urologic malignancy in the United States. Muscle invasive BLCA is a morbid and expensive disease to treat, thus increased molecular understanding is necessary. BLCA has recently been characterized by their distinct expression of luminal and basal genes, which could be used to predict key clinical features such as disease progression and overall survival. Transcriptionally, FOXA1, GATA3, and PPARg have been shown to be essential for luminal subtype-specific gene regulation and subtype switching, while TP63, STAT3, and TFAP2 family members are critical for regulation of basal subtype specific genes. Despite these advances, the underlying epigenetic mechanisms and 3D chromatin architecture responsible for subtype-specific regulation in bladder cancer remains unknown. Here, we determined the genome-wide transcriptome, enhancer landscape, and transcription factor binding profiles (FOXA1 and GATA3) in luminal and basal subtypes of bladder cancers. Furthermore, we report the first-ever mapping of genome-wide chromatin interactions by Hi-C in both bladder cancer cell lines and primary patient tumors. We show that subtype-specific transcription is accompanied by specific open chromatin and epigenomic marks, at least partially driven by distinct TF binding at distal-enhancers of luminal and basal bladder cancers. Finally, we identified a novel clinically relevant transcriptional factor, Neuronal PAS Domain Protein 2 (NPAS2), in luminal bladder cancers that regulates other subtype-specific genes and influences cancer cell proliferation and migration. In summary, our work identifies a subtype-specific epigenomic and 3D genome structure in urinary bladder cancers and suggests a novel link between the circadian TF NPAS2 and a clinical bladder cancer subtype. Citation Format: Tejaswi Tejaswi, Baozhen Zhang, Qixuan Wang, Ye Hou, Qiushi Jin, Jie Xu, Hongbo Yang, Tingting Liu, David J. DeGraff, Feng Yue. Subtype-specific epigenomic landscape and 3D genome structure in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2189.

Subtype-associated epigenomic landscape and 3D genome structure in bladder cancer

Muscle-invasive bladder cancers are characterized by their distinct expression of luminal and basal genes, which could be used to predict key clinical features such as disease progression and overall survival. Transcriptionally, FOXA1, GATA3, and PPARG are shown to be essential for luminal subtype-specific gene regulation and subtype switching, while TP63, STAT3, and TFAP2 family members are critical for regulation of basal subtype-specific genes. Despite these advances, the underlying epigenetic mechanisms and 3D chromatin architecture responsible for subtype-specific regulation in bladder cancer remain unknown.ResultWe determine the genome-wide transcriptome, enhancer landscape, and transcription factor binding profiles of FOXA1 and GATA3 in luminal and basal subtypes of bladder cancer. Furthermore, we report the first-ever mapping of genome-wide chromatin interactions by Hi-C in both bladder cancer cell lines and primary patient tumors. We show that subtype-specific transcription is accompanied by specific open chromatin and epigenomic marks, at least partially driven by distinct transcription factor binding at distal enhancers of luminal and basal bladder cancers. Finally, we identify a novel clinically relevant transcription factor, Neuronal PAS Domain Protein 2 (NPAS2), in luminal bladder cancers that regulates other subtype-specific genes and influences cancer cell proliferation and migration.ConclusionIn summary, our work identifies unique epigenomic signatures and 3D genome structures in luminal and basal urinary bladder cancers and suggests a novel link between the circadian transcription factor NPAS2 and a clinical bladder cancer subtype.

Systematic analysis of enhancer regulatory circuit perturbation driven by copy number variations in malignant glioma.

Background: Enhancers are emerging regulatory regions controlling gene expression in diverse cancer types. However, the functions of enhancer regulatory circuit perturbations driven by copy number variations (CNVs) in malignant glioma are unclear. Therefore, we aimed to investigate the comprehensive enhancer regulatory perturbation and identify potential biomarkers in glioma.Results: We performed a meta-analysis of the enhancer centered regulatory circuit perturbations in 683 gliomas by integrating CNVs, gene expression, and transcription factors (TFs) binding. We found widespread CNVs of enhancers during glioma progression, and CNVs were associated with the perturbations of enhancer activities. In particular, the degree of perturbations for amplified enhancers was much greater accompanied by the glioma malignant progression. In addition, CNVs and enhancers cooperatively regulated the expressions of cancer-related genes. Genome-wide TF binding profiles revealed that enhancers were pervasively regulated by TFs. A network-based analysis of TF-enhancer-gene regulatory circuits revealed a core TF-gene module (58 interactions including seven genes and 14 TFs) that was associated survival of patients with glioma (p < 0.001). Finally, we validated this prognosis-associated TF-gene regulatory module in an independent cohort. In summary, our analyses provided new molecular insights for enhancer-centered transcriptional perturbation in glioma therapy.Conclusion: Integrative analysis revealed enhancer regulatory perturbations in glioma and also identified a network module that was associated with patient survival, thereby providing novel insights into enhancer-centered cancer therapy.

BICORN: An R package for integrative inference of de novo cis-regulatory modules

Genome-wide transcription factor (TF) binding signal analyses reveal co-localization of TF binding sites, based on which cis-regulatory modules (CRMs) can be inferred. CRMs play a key role in understanding the cooperation of multiple TFs under specific conditions. However, the functions of CRMs and their effects on nearby gene transcription are highly dynamic and context-specific and therefore are challenging to characterize. BICORN (Bayesian Inference of COoperative Regulatory Network) builds a hierarchical Bayesian model and infers context-specific CRMs based on TF-gene binding events and gene expression data for a particular cell type. BICORN automatically searches for a list of candidate CRMs based on the input TF bindings at regulatory regions associated with genes of interest. Applying Gibbs sampling, BICORN iteratively estimates model parameters of CRMs, TF activities, and corresponding regulation on gene transcription, which it models as a sparse network of functional CRMs regulating target genes. The BICORN package is implemented in R (version 3.4 or later) and is publicly available on the CRAN server at https://cran.r-project.org/web/packages/BICORN/index.html.

Intricate Genetic Programs Controlling Dormancy in Mycobacterium tuberculosis

SUMMARYMycobacterium tuberculosis (MTB) displays the remarkable ability to transition in and out of dormancy, a hallmark of the pathogen’s capacity to evade the immune system and exploit susceptible individuals. Uncovering the gene regulatory programs that underlie the phenotypic shifts in MTB during disease latency and reactivation has posed a challenge. We develop an experimental system to precisely control dissolved oxygen levels in MTB cultures in order to capture the transcriptional events that unfold as MTB transitions into and out of hypoxia-induced dormancy. Using a comprehensive genome-wide transcription factor binding map and insights from network topology analysis, we identify regulatory circuits that deterministically drive sequential transitions across six transcriptionally and functionally distinct states encompassing more than three-fifths of the MTB genome. The architecture of the genetic programs explains the transcriptional dynamics underlying synchronous entry of cells into a dormant state that is primed to infect the host upon encountering favorable conditions.

GATA Factor-Mediated Gene Regulation in Human Erythropoiesis.

SummaryErythroid commitment and differentiation are regulated by the coordinated action of a host of transcription factors, including GATA2 and GATA1. Here, we explored GATA-mediated transcriptional regulation through the integrative analysis of gene expression, chromatin modifications, and GATA factors' binding in human multipotent hematopoietic stem/progenitor cells, early erythroid progenitors, and late precursors. A progressive loss of H3K27 acetylation and a diminished usage of active enhancers and super-enhancers were observed during erythroid commitment and differentiation. GATA factors mediate transcriptional changes through a stage-specific interplay with regulatory elements: GATA1 binds different sets of regulatory elements in erythroid progenitors and precursors and controls the transcription of distinct genes during commitment and differentiation. Importantly, our results highlight a pivotal role of promoters in determining the transcriptional program activated upon erythroid differentiation. Finally, we demonstrated that GATA1 binding to a stage-specific super-enhancer sustains the expression of the KIT receptor in human erythroid progenitors.

A Comprehensive FXR Signaling Atlas Derived from Pooled ChIP-seq Data.

ChIP-seq is a method to identify genome-wide transcription factor (TF) binding sites. The TF FXR is a nuclear receptor that controls gene regulation of different metabolic pathways in the liver. To re-analyze, standardize and combine all publicly available FXR ChIP-seq data sets to create a global FXR signaling atlas. All data sets were (re-)analyzed in a standardized manner and compared on every relevant level from raw reads to affected functional pathways. Public FXR data sets were available for mouse, rat and primary human hepatocytes in different treatment conditions. Standardized re-analysis shows that the data sets are surprisingly heterogeneous concerning baseline quality criteria. Combining different data sets increased the depth of analysis and allowed to recover more peaks and functional pathways. Published single FXR ChIP-seq data sets do not cover the full spectrum of FXR signaling. Combining different data sets and creating a "FXR super-signaling atlas" enhances understanding of FXR signaling capacities.

Genome-Wide Transcription Factor Binding in Leaves from C3 and C4 Grasses.

The majority of plants use C3 photosynthesis, but over 60 independent lineages of angiosperms have evolved the C4 pathway. In most C4 species, photosynthesis gene expression is compartmented between mesophyll and bundle-sheath cells. We performed DNaseI sequencing to identify genome-wide profiles of transcription factor binding in leaves of the C4 grasses Zea mays, Sorghum bicolor, and Setaria italica as well as C3 Brachypodium distachyon In C4 species, while bundle-sheath strands and whole leaves shared similarity in the broad regions of DNA accessible to transcription factors, the short sequences bound varied. Transcription factor binding was prevalent in gene bodies as well as promoters, and many of these sites could represent duons that influence gene regulation in addition to amino acid sequence. Although globally there was little correlation between any individual DNaseI footprint and cell-specific gene expression, within individual species transcription factor binding to the same motifs in multiple genes provided evidence for shared mechanisms governing C4 photosynthesis gene expression. Furthermore, interspecific comparisons identified a small number of highly conserved transcription factor binding sites associated with leaves from species that diverged around 60 million years ago. These data therefore provide insight into the architecture associated with C4 photosynthesis gene expression in particular and characteristics of transcription factor binding in cereal crops in general.

FUNCTIONAL DISSECTION OF TRANSCRIPTION FACTOR NETWORKS GOVERNING HAEMATOPOIETIC PROGENITOR STATES

Maintenance of the adult haematopoietic system requires carefully balanced cell fate decisions at the level of multipotent progenitor cells. These decisions are controlled by complex transcription factor (TF) networks. While some TF-target connections have been experimentally established, they are mostly inferred from correlations of either gene expression (RNA-Seq) or genome-wide binding events (ChIP-Seq). This limits our understanding and prevents informed manipulation of cell states. To address these issues, we used CRISPR/Cas9 system and small scale RNA-Seq to experimentally assay gene regulatory functions of 38 TFs in the Hoxb8-FL cellular system, which captures in vitro a lymphoid-primed multipotent progenitor (LMPP) like state. Our RNA-Seq analysis following each TF knockout revealed >15,000 functional activating/inhibitory relationships across >7000 genes. Network analysis augmented with genome-wide TF binding information predicted direct interactions, regulatory elements and putative new circuits maintaining the multipotent state. Among many others, this included a novel interplay between co-existing Ebf1 and Gata3 regulating expression of lymphoid and B-cell specific genes. To gain further insights into how TFs regulate cell state transitions, we developed a new computational method that visualises perturbation effects in the context of single cell expression landscapes. Importantly, this avoids using externally curated ontology databases and instead directly employs human or mouse expression data. In the current study, the analysis revealed how each TF is linked with diverse differentiation programs. In addition to revealing known functions, we also identified previously unrecognized associations, such as Meis1 suppression of the monocytic programme. Studies are underway to validate new circuits and their influence on differentiation in primary cells.