Antagonism of B cell enhancer networks by STAT5 drives leukemia and poor patient survival.

The transcription factor STAT5 has a critical role in B cell acute lymphoblastic leukemia (B-ALL). How STAT5 mediates this effect is unclear. Here we found that activation of STAT5 worked together with defects in signaling components of the precursor to the B cell antigen receptor (pre-BCR), including defects in BLNK, BTK, PKCß, NF-κB1 and IKAROS, to initiate B-ALL. STAT5 antagonized the transcription factors NF-κB and IKAROS by opposing regulation of shared target genes. Super-enhancers showed enrichment for STAT5 binding and were associated with an opposing network of transcription factors, including PAX5, EBF1, PU.1, IRF4 and IKAROS. Patients with a high ratio of active STAT5 to NF-κB or IKAROS had more-aggressive disease. Our studies indicate that an imbalance of two opposing transcriptional programs drives B-ALL and suggest that restoring the balance of these pathways might inhibit B-ALL.

Selective regulation of lymphopoiesis and leukemogenesis by individual zinc fingers of Ikaros.

C2H2 zinc fingers are found in several key transcriptional regulators in the immune system. However, these proteins usually contain more fingers than are needed for sequence-specific DNA binding, which suggests that different fingers regulate different genes and functions. Here we found that mice lacking finger 1 or finger 4 of Ikaros exhibited distinct subsets of the hematological defects of Ikaros-null mice. Most notably, the two fingers controlled different stages of lymphopoiesis, and finger 4 was selectively required for tumor suppression. The distinct defects support the hypothesis that only a small number of genes that are targets of Ikaros are critical for each of its biological functions. The subcategorization of functions and target genes by mutagenesis of individual zinc fingers will facilitate efforts to understand how zinc-finger transcription factors regulate development, immunity and disease.

Distinct regulatory networks control toxin gene expression in elapid and viperid snakes.

BACKGROUND: Venom systems are ideal models to study genetic regulatory mechanisms that underpin evolutionary novelty. Snake venom glands are thought to share a common origin, but there are major distinctions between venom toxins from the medically significant snake families Elapidae and Viperidae, and toxin gene regulatory investigations in elapid snakes have been limited. Here, we used high-throughput RNA-sequencing to profile gene expression and microRNAs between active (milked) and resting (unmilked) venom glands in an elapid (Eastern Brown Snake, Pseudonaja textilis), in addition to comparative genomics, to identify cis- and trans-acting regulation of venom production in an elapid in comparison to viperids (Crotalus viridis and C. tigris). RESULTS: Although there is conservation in high-level mechanistic pathways regulating venom production (unfolded protein response, Notch signaling and cholesterol homeostasis), there are differences in the regulation of histone methylation enzymes, transcription factors, and microRNAs in venom glands from these two snake families. Histone methyltransferases and transcription factor (TF) specificity protein 1 (Sp1) were highly upregulated in the milked elapid venom gland in comparison to the viperids, whereas nuclear factor I (NFI) TFs were upregulated after viperid venom milking. Sp1 and NFI cis-regulatory elements were common to toxin gene promoter regions, but many unique elements were also present between elapid and viperid toxins. The presence of Sp1 binding sites across multiple elapid toxin gene promoter regions that have been experimentally determined to regulate expression, in addition to upregulation of Sp1 after venom milking, suggests this transcription factor is involved in elapid toxin expression. microRNA profiles were distinctive between milked and unmilked venom glands for both snake families, and microRNAs were predicted to target a diversity of toxin transcripts in the elapid P. textilis venom gland, but only snake venom metalloproteinase transcripts in the viperid C. viridis venom gland. These results suggest differences in toxin gene posttranscriptional regulation between the elapid P. textilis and viperid C. viridis. CONCLUSIONS: Our comparative transcriptomic and genomic analyses between toxin genes and isoforms in elapid and viperid snakes suggests independent toxin regulation between these two snake families, demonstrating multiple different regulatory mechanisms underpin a venomous phenotype.

Bromodomain Proteins Epigenetically Regulate the Mitotically Associated lncRNA MANCR in Triple Negative Breast Cancer Cells.

Long non-coding RNA (lncRNA)-mediated control of gene expression contributes to regulation of biological processes that include proliferation and phenotype, as well as compromised expression of genes that are functionally linked to cancer initiation and tumor progression. lncRNAs have emerged as novel targets and biomarkers in breast cancer. We have shown that mitotically associated lncRNA MANCR is expressed in triple-negative breast cancer (TNBC) cells and that it serves a critical role in promoting genome stability and survival in aggressive breast cancer cells. Using an siRNA strategy, we selectively depleted BRD2, BRD3, and BRD4, singly and in combination, to establish which bromodomain proteins regulate MANCR expression in TNBC cells. Our findings were confirmed by using in situ hybridization combined with immunofluorescence analysis that revealed BRD4, either alone or with BRD2 and BRD3, can support MANCR regulation of TNBC cells. Here we provide evidence for MANCR-responsive epigenetic control of super enhancers by histone modifications that are required for gene transcription to support cell survival and expression of the epithelial tumor phenotype in triple negative breast cancer cells.

Transcriptome analysis of primary adult B-cell lineage acute lymphoblastic leukemia identifies pathogenic variants and gene fusions, and predicts subtypes for in depth molecular diagnosis.

BACKGROUND: B-cell acute lymphoblastic leukemia (B-ALL) is classified into subgroups based on known driver oncogenes and molecular lesions, including translocations and recurrent mutations. However, the current diagnostic tests do not identify subtypes or oncogenic lesions for all B-ALL samples, creating a heterogeneous B-ALL group of unknown subtypes. METHODS: We sorted primary adult B-ALL cells and performed transcriptome analysis by bulk RNA sequencing (RNA-seq). RESULTS: Transcriptomic analysis of an adult B-ALL cohort allowed the classification of four patient samples with subtypes that were not previously revealed by standard gene panels. The leukemia of two patients were of the DUX4 subtype and two were CRLF2+ Ph-like B-ALL. Furthermore, single nucleotide variant analysis detected the oncogenic NRAS-G12D, KRAS-G12D, and KRAS-G13D mutations in three of the patient samples, presenting targetable mutations. Additional oncogenic variants and gene fusions were uncovered, as well as multiple variants in the PDE4DIP gene across five of the patient samples. CONCLUSION: We demonstrate that RNA-seq is an effective tool for precision medicine in B-ALL by providing comprehensive molecular profiling of leukemia cells, identifying subtype and oncogenic lesions, and stratifying patients for appropriate therapy.

Thyroid hormone dependent transcriptional programming by TRß requires SWI/SNF chromatin remodelers.

Transcriptional regulation in response to thyroid hormone (3,5,3'-triiodo-l-thyronine, T3) is a dynamic and cell-type specific process that maintains cellular homeostasis and identity in all tissues. However, our understanding of the mechanisms of thyroid hormone receptor (TR) actions at the molecular level are actively being refined. We used an integrated genomics approach to profile and characterize the cistrome of TRß, map changes in chromatin accessibility, and capture the transcriptomic changes in response to T3 in normal human thyroid cells. There are significant shifts in TRß genomic occupancy in response to T3, which are associated with differential chromatin accessibility, and differential recruitment of SWI/SNF chromatin remodelers. We further demonstrate selective recruitment of BAF and PBAF SWI/SNF complexes to TRß binding sites, revealing novel differential functions in regulating chromatin accessibility and gene expression. Our findings highlight three distinct modes of TRß interaction with chromatin and coordination of coregulator activity.

peaksat: an R package for ChIP-seq peak saturation analysis.

BACKGROUND: Epigenomic profiling assays such as ChIP-seq have been widely used to map the genome-wide enrichment profiles of chromatin-associated proteins and posttranslational histone modifications. Sequencing depth is a key parameter in experimental design and quality control. However, due to variable sequencing depth requirements across experimental conditions, it can be challenging to determine optimal sequencing depth, particularly for projects involving multiple targets or cell types. RESULTS: We developed the peaksat R package to provide target read depth estimates for epigenomic experiments based on the analysis of peak saturation curves. We applied peaksat to establish the distinctive read depth requirements for ChIP-seq studies of histone modifications in different cell lines. Using peaksat, we were able to estimate the target read depth required per library to obtain high-quality peak calls for downstream analysis. In addition, peaksat was applied to other sequence-enrichment methods including CUT&RUN and ATAC-seq. CONCLUSION: peaksat addresses a need for researchers to make informed decisions about whether their sequencing data has been generated to an adequate depth and subsequently sufficient meaningful peaks, and failing that, how many more reads would be required per library. peaksat is applicable to other sequence-based methods that include calling peaks in their analysis.

Spatiotemporal Epigenetic Control of the Histone Gene Chromatin Landscape during the Cell Cycle.

Higher-order genomic organization supports the activation of histone genes in response to cell cycle regulatory cues that epigenetically mediates stringent control of transcription at the G1/S-phase transition. Histone locus bodies (HLBs) are dynamic, non-membranous, phase-separated nuclear domains where the regulatory machinery for histone gene expression is organized and assembled to support spatiotemporal epigenetic control of histone genes. HLBs provide molecular hubs that support synthesis and processing of DNA replication-dependent histone mRNAs. These regulatory microenvironments support long-range genomic interactions among non-contiguous histone genes within a single topologically associating domain (TAD). HLBs respond to activation of the cyclin E/CDK2/NPAT/HINFP pathway at the G1/S transition. HINFP and its coactivator NPAT form a complex within HLBs that controls histone mRNA transcription to support histone protein synthesis and packaging of newly replicated DNA. Loss of HINFP compromises H4 gene expression and chromatin formation, which may result in DNA damage and impede cell cycle progression. HLBs provide a paradigm for higher-order genomic organization of a subnuclear domain that executes an obligatory cell cycle-controlled function in response to cyclin E/CDK2 signaling. Understanding the coordinately and spatiotemporally organized regulatory programs in focally defined nuclear domains provides insight into molecular infrastructure for responsiveness to cell signaling pathways that mediate biological control of growth, differentiation phenotype, and are compromised in cancer.

Common tumor-suppressive signaling of thyroid hormone receptor beta in breast and thyroid cancer cells.

The thyroid hormone receptor beta (TRß) is a tumor suppressor in multiple types of solid tumors, most prominently in breast and thyroid cancer. An increased understanding of the molecular mechanisms by which TRß abrogates tumorigenesis will aid in understanding the core tumor-suppressive functions of TRß. Here, we restored TRß expression in the MDA-MB-468 basal-like breast cancer cell line and perform RNA-sequencing to determine the TRß-mediated changes in gene expression and associated signaling pathways. The TRß expressing MDA-MB-468 cells exhibit a more epithelial character as determined by principle component analysis-based iterative PAM50 subtyping score and through reduced expression of mesenchymal cytokeratins. The epithelial to mesenchymal transition pathway is also significantly reduced. The MDA-MB-468 data set was further compared with RNA sequencing results from TRß expressing thyroid cancer cell line SW1736 to determine which genes are TRß correspondingly regulated across both cell types. Several pathways including lipid metabolism and chromatin remodeling processes were observed to be altered in the shared gene set. These data provide novel insights into the molecular mechanisms by which TRß suppresses breast tumorigenesis.

Non-Coding RNA Signatures of B-Cell Acute Lymphoblastic Leukemia.

Non-coding RNAs (ncRNAs) comprise a diverse class of non-protein coding transcripts that regulate critical cellular processes associated with cancer. Advances in RNA-sequencing (RNA-Seq) have led to the characterization of non-coding RNA expression across different types of human cancers. Through comprehensive RNA-Seq profiling, a growing number of studies demonstrate that ncRNAs, including long non-coding RNA (lncRNAs) and microRNAs (miRNA), play central roles in progenitor B-cell acute lymphoblastic leukemia (B-ALL) pathogenesis. Furthermore, due to their central roles in cellular homeostasis and their potential as biomarkers, the study of ncRNAs continues to provide new insight into the molecular mechanisms of B-ALL. This article reviews the ncRNA signatures reported for all B-ALL subtypes, focusing on technological developments in transcriptome profiling and recently discovered examples of ncRNAs with biologic and therapeutic relevance in B-ALL.

RUNX1 and RUNX2 transcription factors function in opposing roles to regulate breast cancer stem cells.

Breast cancer stem cells (BCSCs) are competent to initiate tumor formation and growth and refractory to conventional therapies. Consequently BCSCs are implicated in tumor recurrence. Many signaling cascades associated with BCSCs are critical for epithelial-to-mesenchymal transition (EMT). We developed a model system to mechanistically examine BCSCs in basal-like breast cancer using MCF10AT1 FACS sorted for CD24 (negative/low in BCSCs) and CD44 (positive/high in BCSCs). Ingenuity Pathway Analysis comparing RNA-seq on the CD24-/low versus CD24+/high MCF10AT1 indicates that the top activated upstream regulators include TWIST1, TGFß1, OCT4, and other factors known to be increased in BCSCs and during EMT. The top inhibited upstream regulators include ESR1, TP63, and FAS. Consistent with our results, many genes previously demonstrated to be regulated by RUNX factors are altered in BCSCs. The RUNX2 interaction network is the top significant pathway altered between CD24-/low and CD24+/high MCF10AT1. RUNX1 is higher in expression at the RNA level than RUNX2. RUNX3 is not expressed. While, human-specific quantitative polymerase chain reaction primers demonstrate that RUNX1 and CDH1 decrease in human MCF10CA1a cells that have grown tumors within the murine mammary fat pad microenvironment, RUNX2 and VIM increase. Treatment with an inhibitor of RUNX binding to CBFß for 5 days followed by a 7-day recovery period results in EMT suggesting that loss of RUNX1, rather than increase in RUNX2, is a driver of EMT in early stage breast cancer. Increased understanding of RUNX regulation on BCSCs and EMT will provide novel insight into therapeutic strategies to prevent recurrence.

The CCCTC Binding Factor, CTRL2, Modulates Heterochromatin Deposition and the Establishment of Herpes Simplex Virus 1 Latency In Vivo.

The cellular insulator protein CTCF plays a role in herpes simplex virus 1 (HSV-1) latency through the establishment and regulation of chromatin boundaries. We previously found that the CTRL2 regulatory element downstream from the latency-associated transcript (LAT) enhancer was bound by CTCF during latency and underwent CTCF eviction at early times postreactivation in mice latently infected with 17syn+ virus. We also showed that CTRL2 was a functional enhancer-blocking insulator in both epithelial and neuronal cell lines. We hypothesized that CTRL2 played a direct role in silencing lytic gene expression during the establishment of HSV-1 latency. To test this hypothesis, we used a recombinant virus with a 135-bp deletion spanning only the core CTRL2 insulator domain (ΔCTRL2) in the 17syn+ background. Deletion of CTRL2 resulted in restricted viral replication in epithelial cells but not neuronal cells. Following ocular infection, mouse survival decreased in the ΔCTRL2-infected cohort, and we found a significant decrease in the number of viral genomes in mouse trigeminal ganglia (TG) infected with ΔCTRL2, indicating that the CTRL2 insulator was required for the efficient establishment of latency. Immediate early (IE) gene expression significantly increased in the number of ganglia infected with ΔCTRL2 by 31 days postinfection relative to the level with 17syn+ infection, indicating that deletion of the CTRL2 insulator disrupted the organization of chromatin domains during HSV-1 latency. Finally, chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) analyses of TG from ΔCTRL2-infected mice confirmed that the distribution of the repressive H3K27me3 (histone H3 trimethylated at K27) mark on the ΔCTRL2 recombinant genomes was altered compared to that of the wild type, indicating that the CTRL2 site modulates the repression of IE genes during latency.IMPORTANCE It is becoming increasingly clear that chromatin insulators play a key role in the transcriptional control of DNA viruses. The gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) utilize chromatin insulators to order protein recruitment and dictate the formation of three-dimensional DNA loops that spatially control transcription and latency. The contribution of chromatin insulators in alphaherpesvirus transcriptional control is less well understood. The work presented here begins to bridge that gap in knowledge by showing how one insulator site in HSV-1 modulates lytic gene transcription and heterochromatin deposition as the HSV-1 genome establishes latency.

RUNX1-dependent mechanisms in biological control and dysregulation in cancer.

The RUNX1 transcription factor has recently been shown to be obligatory for normal development. RUNX1 controls the expression of genes essential for proper development in many cell lineages and tissues including blood, bone, cartilage, hair follicles, and mammary glands. Compromised RUNX1 regulation is associated with many cancers. In this review, we highlight evidence for RUNX1 control in both invertebrate and mammalian development and recent novel findings of perturbed RUNX1 control in breast cancer that has implications for other solid tumors. As RUNX1 is essential for definitive hematopoiesis, RUNX1 mutations in hematopoietic lineage cells have been implicated in the etiology of several leukemias. Studies of solid tumors have revealed a context-dependent function for RUNX1 either as an oncogene or a tumor suppressor. These RUNX1 functions have been reported for breast, prostate, lung, and skin cancers that are related to cancer subtypes and different stages of tumor development. Growing evidence suggests that RUNX1 suppresses aggressiveness in most breast cancer subtypes particularly in the early stage of tumorigenesis. Several studies have identified RUNX1 suppression of the breast cancer epithelial-to-mesenchymal transition. Most recently, RUNX1 repression of cancer stem cells and tumorsphere formation was reported for breast cancer. It is anticipated that these new discoveries of the context-dependent diversity of RUNX1 functions will lead to innovative therapeutic strategies for the intervention of cancer and other abnormalities of normal tissues.

Three-dimensional analysis reveals altered chromatin interaction by enhancer inhibitors harbors TCF7L2-regulated cancer gene signature.

Distal regulatory elements influence the activity of gene promoters through chromatin looping. Chromosome conformation capture (3C) methods permit identification of chromatin contacts across different regions of the genome. However, due to limitations in the resolution of these methods, the detection of functional chromatin interactions remains a challenge. In the current study, we employ an integrated approach to define and characterize the functional chromatin contacts of human pancreatic cancer cells. We applied tethered chromatin capture to define classes of chromatin domains on a genome-wide scale. We identified three types of structural domains (topologically associated, boundary, and gap) and investigated the functional relationships of these domains with respect to chromatin state and gene expression. We uncovered six distinct sub-domains associated with epigenetic states. Interestingly, specific epigenetically active domains are sensitive to treatment with histone acetyltransferase (HAT) inhibitors and decrease in H3K27 acetylation levels. To examine whether the subdomains that change upon drug treatment are functionally linked to transcription factor regulation, we compared TCF7L2 chromatin binding and gene regulation to HAT inhibition. We identified a subset of coding RNA genes that together can stratify pancreatic cancer patients into distinct survival groups. Overall, this study describes a process to evaluate the functional features of chromosome architecture and reveals the impact of epigenetic inhibitors on chromosome architecture and identifies genes that may provide insight into disease outcome.

Varicella Zoster Virus Induces Nuclear Translocation of the Neurokinin-1 Receptor, Promoting Lamellipodia Formation and Viral Spread in Spinal Astrocytes.

Background: Varicella zoster virus (VZV) can present as a myelopathy with spinal astrocyte infection. Recent studies support a role for the neurokinin-1 receptor (NK-1R) in virus infections, as well as for cytoskeletal alterations that may promote viral spread. Thus, we examined the role of NK-1R in VZV-infected primary human spinal astrocytes (HA-sps) to shed light on the pathogenesis of VZV myelopathy. Methods: Mock- and VZV-infected HA-sps were examined for substance P (subP) production, NK-1R localization, morphological changes, and viral spread in the presence or absence of the NK-1R antagonists aprepitant and rolapitant. Results: VZV infection of HA-sps induced nuclear localization of full-length and truncated NK-1R in the absence of the endogenous ligand, subP, and was associated with extensive lamellipodia formation and viral spread that was inhibited by NK-1R antagonists. Conclusions: We have identified a novel, subP-independent, proviral function of nuclear NK-1R associated with lamellipodia formation and viral spread that is distinct from subP-induced NK-1R cell membrane/cytoplasmic localization without lamellipodia formation. These results suggest that binding of a putative viral ligand to NK-1R produces a dramatically different NK-1R downstream effect than binding of subP. Finally, the NK-1R antagonists aprepitant and rolapitant provide promising alternatives to nucleoside analogs in treating VZV infections, including myelopathy.

Intranuclear and higher-order chromatin organization of the major histone gene cluster in breast cancer.

Alterations in nuclear morphology are common in cancer progression. However, the degree to which gross morphological abnormalities translate into compromised higher-order chromatin organization is poorly understood. To explore the functional links between gene expression and chromatin structure in breast cancer, we performed RNA-seq gene expression analysis on the basal breast cancer progression model based on human MCF10A cells. Positional gene enrichment identified the major histone gene cluster at chromosome 6p22 as one of the most significantly upregulated (and not amplified) clusters of genes from the normal-like MCF10A to premalignant MCF10AT1 and metastatic MCF10CA1a cells. This cluster is subdivided into three sub-clusters of histone genes that are organized into hierarchical topologically associating domains (TADs). Interestingly, the sub-clusters of histone genes are located at TAD boundaries and interact more frequently with each other than the regions in-between them, suggesting that the histone sub-clusters form an active chromatin hub. The anchor sites of loops within this hub are occupied by CTCF, a known chromatin organizer. These histone genes are transcribed and processed at a specific sub-nuclear microenvironment termed the major histone locus body (HLB). While the overall chromatin structure of the major HLB is maintained across breast cancer progression, we detected alterations in its structure that may relate to gene expression. Importantly, breast tumor specimens also exhibit a coordinate pattern of upregulation across the major histone gene cluster. Our results provide a novel insight into the connection between the higher-order chromatin organization of the major HLB and its regulation during breast cancer progression.

Adaptive evolution of distinct prey-specific toxin genes in rear-fanged snake venom.

Venom proteins evolve rapidly, and as a trophic adaptation are excellent models for predator-prey evolutionary studies. The key to a deeper understanding of venom evolution is an integrated approach, combining prey assays with analysis of venom gene expression and venom phenotype. Here, we use such an approach to study venom evolution in the Amazon puffing snake, Spilotes sulphureus, a generalist feeder. We identify two novel three-finger toxins: sulditoxin and sulmotoxin 1. These new toxins are not only two of the most abundant venom proteins, but are also functionally intriguing, displaying distinct prey-specific toxicities. Sulditoxin is highly toxic towards lizard prey, but is non-toxic towards mammalian prey, even at greater than 22-fold higher dosage. By contrast, sulmotoxin 1 exhibits the reverse trend. Furthermore, evolutionary analysis and structural modelling show highest sequence variability in the central loop of these proteins, probably driving taxon-specific toxicity. This is, to our knowledge, the first case in which a bimodal and contrasting pattern of toxicity has been shown for proteins in the venom of a single snake in relation to diet. Our study is an example of how toxin gene neofunctionalization can result in a venom system dominated by one protein superfamily and still exhibit flexibility in prey capture efficacy.

Targeted Genome Sequencing Reveals Varicella-Zoster Virus Open Reading Frame 12 Deletion.

The neurotropic herpesvirus varicella-zoster virus (VZV) establishes a lifelong latent infection in humans following primary infection. The low abundance of VZV nucleic acids in human neurons has hindered an understanding of the mechanisms that regulate viral gene transcription during latency. To overcome this critical barrier, we optimized a targeted capture protocol to enrich VZV DNA and cDNA prior to whole-genome/transcriptome sequence analysis. Since the VZV genome is remarkably stable, it was surprising to detect that VZV32, a VZV laboratory strain with no discernible growth defect in tissue culture, contained a 2,158-bp deletion in open reading frame (ORF) 12. Consequently, ORF 12 and 13 protein expression was abolished and Akt phosphorylation was inhibited. The discovery of the ORF 12 deletion, revealed through targeted genome sequencing analysis, points to the need to authenticate the VZV genome when the virus is propagated in tissue culture.IMPORTANCE Viruses isolated from clinical samples often undergo genetic modifications when cultured in the laboratory. Historically, VZV is among the most genetically stable herpesviruses, a notion supported by more than 60 complete genome sequences from multiple isolates and following multiple in vitro passages. However, application of enrichment protocols to targeted genome sequencing revealed the unexpected deletion of a significant portion of VZV ORF 12 following propagation in cultured human fibroblast cells. While the enrichment protocol did not introduce bias in either the virus genome or transcriptome, the findings indicate the need for authentication of VZV by sequencing when the virus is propagated in tissue culture.

Architecture of the human regulatory network derived from ENCODE data.

Transcription factors bind in a combinatorial fashion to specify the on-and-off states of genes; the ensemble of these binding events forms a regulatory network, constituting the wiring diagram for a cell. To examine the principles of the human transcriptional regulatory network, we determined the genomic binding information of 119 transcription-related factors in over 450 distinct experiments. We found the combinatorial, co-association of transcription factors to be highly context specific: distinct combinations of factors bind at specific genomic locations. In particular, there are significant differences in the binding proximal and distal to genes. We organized all the transcription factor binding into a hierarchy and integrated it with other genomic information (for example, microRNA regulation), forming a dense meta-network. Factors at different levels have different properties; for instance, top-level transcription factors more strongly influence expression and middle-level ones co-regulate targets to mitigate information-flow bottlenecks. Moreover, these co-regulations give rise to many enriched network motifs (for example, noise-buffering feed-forward loops). Finally, more connected network components are under stronger selection and exhibit a greater degree of allele-specific activity (that is, differential binding to the two parental alleles). The regulatory information obtained in this study will be crucial for interpreting personal genome sequences and understanding basic principles of human biology and disease.

Genome-wide analysis reveals positional-nucleosome-oriented binding pattern of pioneer factor FOXA1.

The compaction of nucleosomal structures creates a barrier for DNA-binding transcription factors (TFs) to access their cognate cis-regulatory elements. Pioneer factors (PFs) such as FOXA1 are able to directly access these cis-targets within compact chromatin. However, how these PFs interplay with nucleosomes remains to be elucidated, and is critical for us to understand the underlying mechanism of gene regulation. Here, we have conducted a computational analysis on a strand-specific paired-end ChIP-exo (termed as ChIP-ePENS) data of FOXA1 in LNCaP cells by our novel algorithm ePEST. We find that FOXA1 chromatin binding occurs via four distinct border modes (or footprint boundary patterns), with a preferential footprint boundary patterns relative to FOXA1 motif orientation. In addition, from this analysis three fundamental nucleotide positions (oG, oS and oH) emerged as major determinants for blocking exo-digestion and forming these four distinct border modes. By integrating histone MNase-seq data, we found an astonishingly consistent, 'well-positioned' configuration occurs between FOXA1 motifs and dyads of nucleosomes genome-wide. We further performed ChIP-seq of eight chromatin remodelers and found an increased occupancy of these remodelers on FOXA1 motifs for all four border modes (or footprint boundary patterns), indicating the full occupancy of FOXA1 complex on the three blocking sites (oG, oS and oH) likely produces an active regulatory status with well-positioned phasing for protein binding events. Together, our results suggest a positional-nucleosome-oriented accessing model for PFs seeking target motifs, in which FOXA1 can examine each underlying DNA nucleotide and is able to sense all potential motifs regardless of whether they face inward or outward from histone octamers along the DNA helix axis.