Exhaustive identification of genome-wide binding events of transcriptional regulators.

Genome-wide binding assays aspire to map the complete binding pattern of gene regulators. Common practice relies on replication-duplicates or triplicates-and high stringency statistics to favor false negatives over false positives. Here we show that duplicates and triplicates of CUT&RUN are not sufficient to discover the entire activity of transcriptional regulators. We introduce ICEBERG (Increased Capture of Enrichment By Exhaustive Replicate aGgregation), a pipeline that harnesses large numbers of CUT&RUN replicates to discover the full set of binding events and chart the line between false positives and false negatives. We employed ICEBERG to map the full set of H3K4me3-marked regions, the targets of the co-factor ß-catenin, and those of the transcription factor TBX3, in human colorectal cancer cells. The ICEBERG datasets allow benchmarking of individual replicates, comparing the performance of peak calling and replication approaches, and expose the arbitrary nature of strategies to identify reproducible peaks. Instead of a static view of genomic targets, ICEBERG establishes a spectrum of detection probabilities across the genome for a given factor, underlying the intrinsic dynamicity of its mechanism of action, and permitting to distinguish frequent from rare regulation events. Finally, ICEBERG discovered instances, undetectable with other approaches, that underlie novel mechanisms of colorectal cancer progression.

A new CUT&RUN low volume-urea (LoV-U) protocol optimized for transcriptional co-factors uncovers Wnt/ß-catenin tissue-specific genomic targets.

Upon WNT/ß-catenin pathway activation, stabilized ß-catenin travels to the nucleus where it associates with the TCF/LEF transcription factors, constitutively bound to genomic Wnt-responsive elements (WREs), to activate target gene transcription. Discovering the binding profile of ß-catenin is therefore required to unambiguously assign direct targets of WNT signaling. Cleavage under targets and release using nuclease (CUT&RUN) has emerged as prime technique for mapping the binding profile of DNA-interacting proteins. Here, we present a modified version of CUT&RUN, named LoV-U (low volume and urea), that enables the robust and reproducible generation of ß-catenin binding profiles, uncovering direct WNT/ß-catenin target genes in human cells, as well as in cells isolated from developing mouse tissues. CUT&RUN-LoV-U outperforms original CUT&RUN when targeting co-factors that do not bind the DNA, can profile all classes of chromatin regulators and is well suited for simultaneous processing of several samples. We believe that the application of our protocol will allow the detection of the complex system of tissue-specific WNT/ß-catenin target genes, together with other non-DNA-binding transcriptional regulators that act downstream of ontogenetically fundamental signaling cascades.

Stress-sensitive dynamics of miRNAs and Elba1 in Drosophila embryogenesis.

Early-life stress can result in life-long effects that impact adult health and disease risk, but little is known about how such programming is established and maintained. Here, we show that such epigenetic memories can be initiated in the Drosophila embryo before the major wave of zygotic transcription, and higher-order chromatin structures are established. An early short heat shock results in elevated levels of maternal miRNA and reduced levels of a subgroup of zygotic genes in stage 5 embryos. Using a Dicer-1 mutant, we show that the stress-induced decrease in one of these genes, the insulator-binding factor Elba1, is dependent on functional miRNA biogenesis. Reduction in Elba1 correlates with the upregulation of early developmental genes and promotes a sustained weakening of heterochromatin in the adult fly as indicated by an increased expression of the PEV wm4h reporter. We propose that maternal miRNAs, retained in response to an early embryonic heat shock, shape the subsequent de novo heterochromatin establishment that occurs during early development via direct or indirect regulation of some of the earliest expressed genes, including Elba1.

Single-cell response to Wnt signaling activation reveals uncoupling of Wnt target gene expression.

Wnt signaling drives nuclear translocation of ß-catenin and its subsequent association with the DNA-bound TCF/LEF transcription factors, which dictate target gene specificity by recognizing Wnt responsive elements across the genome. ß-Catenin target genes are therefore thought to be collectively activated upon Wnt pathway stimulation. However, this appears in contrast with the non-overlapping patterns of Wnt target gene expression in several contexts, including early mammalian embryogenesis. Here we followed Wnt target gene expression in human embryonic stem cells after Wnt pathway stimulation at a single-cell resolution. Cells changed gene expression program over time consistent with three key developmental events: i) loss of pluripotency, ii) induction of Wnt target genes, and iii) mesoderm specification. Contrary to our expectation, not all cells displayed equal amplitude of Wnt target gene activation; rather, they distributed in a continuum from strong to weak responders when ranked based on the expression of the target AXIN2. Moreover, high AXIN2 did not always correspond to elevated expression of other Wnt targets, which were activated in different proportions in individual cells. The uncoupling of Wnt target gene expression was also identified in single cell transcriptomics profiling of other Wnt-responding cell types, including HEK293T, murine developing forelimbs, and human colorectal cancer. Our finding underlines the necessity to identify additional mechanisms that explain the heterogeneity of the Wnt/ß-catenin-mediated transcriptional outputs in single cells.

Transcriptional repression of the oncofetal LIN28B gene by the transcription factor SOX6.

The identification of regulatory networks contributing to fetal/adult gene expression switches is a major challenge in developmental biology and key to understand the aberrant proliferation of cancer cells, which often reactivate fetal oncogenes. One key example is represented by the developmental gene LIN28B, whose aberrant reactivation in adult tissues promotes tumor initiation and progression. Despite the prominent role of LIN28B in development and cancer, the mechanisms of its transcriptional regulation are largely unknown. Here, by using quantitative RT-PCR and single cell RNA sequencing data, we show that in erythropoiesis the expression of the transcription factor SOX6 matched a sharp decline of LIN28B mRNA during human embryo/fetal to adult globin switching. SOX6 overexpression repressed LIN28B not only in a panel of fetal-like erythroid cells (K562, HEL and HUDEP1; ≈92% p < 0.0001, 54% p = 0.0009 and ≈60% p < 0.0001 reduction, respectively), but also in hepatoblastoma HepG2 and neuroblastoma SH-SY5H cells (≈99% p < 0.0001 and ≈59% p < 0.0001 reduction, respectively). SOX6-mediated repression caused downregulation of the LIN28B/Let-7 targets, including MYC and IGF2BP1, and rapidly blocks cell proliferation. Mechanistically, Lin28B repression is accompanied by SOX6 physical binding within its locus, suggesting a direct mechanism of LIN28B downregulation that might contribute to the fetal/adult erythropoietic transition and restrict cancer proliferation.

The CUT&RUN suspect list of problematic regions of the genome.

BACKGROUND: Cleavage Under Targets and Release Using Nuclease (CUT&RUN) is an increasingly popular technique to map genome-wide binding profiles of histone modifications, transcription factors, and co-factors. The ENCODE project and others have compiled blacklists for ChIP-seq which have been widely adopted: these lists contain regions of high and unstructured signal, regardless of cell type or protein target, indicating that these are false positives. While CUT&RUN obtains similar results to ChIP-seq, its biochemistry and subsequent data analyses are different. We found that this results in a CUT&RUN-specific set of undesired high-signal regions. RESULTS: We compile suspect lists based on CUT&RUN data for the human and mouse genomes, identifying regions consistently called as peaks in negative controls. Using published CUT&RUN data from our and other labs, we show that the CUT&RUN suspect regions can persist even when peak calling is performed with SEACR or MACS2 against a negative control and after ENCODE blacklist removal. Moreover, we experimentally validate the CUT&RUN suspect lists by performing reiterative negative control experiments in which no specific protein is targeted, showing that they capture more than 80% of the peaks identified. CONCLUSIONS: We propose that removing these problematic regions can substantially improve peak calling in CUT&RUN experiments, resulting in more reliable datasets.

The time-resolved genomic impact of Wnt/ß-catenin signaling.

Wnt signaling orchestrates gene expression via its effector, ß-catenin. However, it is unknown whether ß-catenin binds its target genomic regions simultaneously and how this impacts chromatin dynamics to modulate cell behavior. Using a combination of time-resolved CUT&RUN against ß-catenin, ATAC-seq, and perturbation assays in different cell types, we show that Wnt/ß-catenin physical targets are tissue-specific, ß-catenin "moves" on different loci over time, and its association to DNA accompanies changing chromatin accessibility landscapes that determine cell behavior. In particular, Wnt/ß-catenin progressively shapes the chromatin of human embryonic stem cells (hESCs) as they undergo mesodermal differentiation, a behavior that we define as "plastic." In HEK293T cells, on the other hand, Wnt/ß-catenin drives a transient chromatin opening, followed by re-establishment of the pre-stimulation state, a response that we define as "elastic." Future experiments shall assess whether other cell communication mechanisms, in addition to Wnt signaling, are ruled by time, cellular idiosyncrasies, and chromatin constraints. A record of this paper's transparent peer review process is included in the supplemental information.