Genomic imprinting is the epigenetic marking of genes that results in parent-of-origin monoallelic expression. Most imprinted domains are associated with differentially DNA methylated regions (DMRs) that originate in the gametes, and are maintained in somatic tissues after fertilization. This allelic methylation profile is associated with a plethora of histone tail modifications that orchestrates higher order chromatin interactions. The mouse chromosome 15 imprinted cluster contains multiple brain-specific maternally expressed transcripts including Ago2, Chrac1, Trappc9 and Kcnk9 and a paternally expressed gene, Peg13. The promoter of Peg13 is methylated on the maternal allele and is the sole DMR within the locus. To determine the extent of imprinting within the human orthologous region on chromosome 8q24, a region associated with autosomal recessive intellectual disability, Birk-Barel mental retardation and dysmorphism syndrome, we have undertaken a systematic analysis of allelic expression and DNA methylation of genes mapping within an approximately 2 Mb region around TRAPPC9.

Self-renewal and pluripotency of the embryonic stem cell (ESC) state are established and maintained by multiple regulatory networks that comprise transcription factors and epigenetic regulators. While much has been learned regarding transcription factors, the function of epigenetic regulators in these networks is less well defined. We conducted a CRISPR-Cas9-mediated loss-of-function genetic screen that identified two epigenetic regulators, TAF5L and TAF6L, components or co-activators of the GNAT-HAT complexes for the mouse ESC (mESC) state. Detailed molecular studies demonstrate that TAF5L/TAF6L transcriptionally activate c-Myc and Oct4 and their corresponding MYC and CORE regulatory networks. Besides, TAF5L/TAF6L predominantly regulate their target genes through H3K9ac deposition and c-MYC recruitment that eventually activate the MYC regulatory network for self-renewal of mESCs. Thus, our findings uncover a role of TAF5L/TAF6L in directing the MYC regulatory network that orchestrates gene expression programs to control self-renewal for the maintenance of mESC state.

Metabolic stress causes activation of the cJun NH2-terminal kinase (JNK) signal transduction pathway. It is established that one consequence of JNK activation is the development of insulin resistance and hepatic steatosis through inhibition of the transcription factor PPARα. Indeed, JNK1/2 deficiency in hepatocytes protects against the development of steatosis, suggesting that JNK inhibition represents a possible treatment for this disease. However, the long-term consequences of JNK inhibition have not been evaluated. Here we demonstrate that hepatic JNK controls bile acid production. We found that hepatic JNK deficiency alters cholesterol metabolism and bile acid synthesis, conjugation, and transport, resulting in cholestasis, increased cholangiocyte proliferation, and intrahepatic cholangiocarcinoma. Gene ablation studies confirmed that PPARα mediated these effects of JNK in hepatocytes. This analysis highlights potential consequences of long-term use of JNK inhibitors for the treatment of metabolic syndrome.

Barrett's esophagus (BE) and gastric intestinal metaplasia are related premalignant conditions in which areas of human stomach epithelium express mixed gastric and intestinal features. Intestinal transcription factors (TFs) are expressed in both conditions, with unclear causal roles and cis-regulatory mechanisms. Ectopic CDX2 reprogrammed isogenic mouse stomach organoid lines to a hybrid stomach-intestinal state transcriptionally similar to clinical metaplasia; squamous esophageal organoids resisted this CDX2-mediated effect. Reprogramming was associated with induced activity at thousands of previously inaccessible intestine-restricted enhancers, where CDX2 occupied DNA directly. HNF4A, a TF recently implicated in BE pathogenesis, induced weaker intestinalization by binding a novel shadow Cdx2 enhancer and hence activating Cdx2 expression. CRISPR/Cas9-mediated germline deletion of that cis-element demonstrated its requirement in Cdx2 induction and in the resulting activation of intestinal genes in stomach cells. dCas9-conjugated KRAB repression mapped this activity to the shadow enhancer's HNF4A binding site. Altogether, we show extensive but selective recruitment of intestinal enhancers by CDX2 in gastric cells and that HNF4A-mediated ectopic CDX2 expression in the stomach occurs through a conserved shadow cis-element. These findings identify mechanisms for TF-driven intestinal metaplasia and a likely pathogenic TF hierarchy.

Reactivation of fetal hemoglobin expression by the downregulation of BCL11A is a promising treatment for β-hemoglobinopathies. A detailed understanding of BCL11A-mediated repression of γ-globin gene (HBG1/2) transcription is lacking, as studies to date used perturbations by shRNA or CRISPR-Cas9 gene editing. We leveraged the dTAG PROTAC degradation platform to acutely deplete BCL11A protein in erythroid cells and examined consequences by nascent transcriptomics, proteomics, chromatin accessibility, and histone profiling. Among 31 genes repressed by BCL11A, HBG1/2 and HBZ show the most abundant and progressive changes in transcription and chromatin accessibility upon BCL11A loss. Transcriptional changes at HBG1/2 were detected in <2 h. Robust HBG1/2 reactivation upon acute BCL11A depletion occurred without the loss of promoter 5-methylcytosine (5mC). Using targeted protein degradation, we establish a hierarchy of gene reactivation at BCL11A targets, in which nascent transcription is followed by increased chromatin accessibility, and both are uncoupled from promoter DNA methylation at the HBG1/2 loci.

We aimed to generate and phenotype a mouse model of foveal hypoplasia, optic nerve decussation defects, and anterior segment dysgenesis (FHONDA), a rare disease associated with mutations in Slc38a8 that causes severe visual alterations similar to albinism without affecting pigmentation.

PDGFRA-expressing mesenchyme supports intestinal stem cells. Stomach epithelia have related niche dependencies, but their enabling mesenchymal cell populations are unknown, in part because previous studies pooled the gastric antrum and corpus. Our high-resolution imaging, transcriptional profiling, and organoid assays identify regional subpopulations and supportive capacities of purified mouse corpus and antral PDGFRA+ cells. Sub-epithelial PDGFRAHi myofibroblasts are principal sources of BMP ligands and two molecularly distinct pools distribute asymmetrically along antral glands but together fail to support epithelial growth in vitro. In contrast, PDGFRALo CD55+ cells strategically positioned beneath gastric glands promote epithelial expansion in the absence of other cells or factors. This population encompasses a small fraction expressing the BMP antagonist Grem1. Although Grem1+ cell ablation in vivo impairs intestinal stem cells, gastric stem cells are spared, implying that CD55+ cell activity in epithelial self-renewal derives from other subpopulations. Our findings shed light on spatial, molecular, and functional organization of gastric mesenchyme and the spectrum of signaling sources for epithelial support.

The CRISPR-Cas system is the newest targeted nuclease for genome engineering. In less than 1 year, the ease, robustness and efficiency of this method have facilitated an immense range of genetic modifications in most model organisms. Full and conditional gene knock-outs, knock-ins, large chromosomal deletions and subtle mutations can be obtained using combinations of clustered regularly interspaced short palindromic repeats (CRISPRs) and DNA donors. In addition, with CRISPR-Cas compounds, multiple genetic modifications can be introduced seamlessly in a single step. CRISPR-Cas not only brings genome engineering capacities to species such as rodents and livestock in which the existing toolbox was already large, but has also enabled precise genetic engineering of organisms with difficult-to-edit genomes such as zebrafish, and of technically challenging species such as non-human primates. The CRISPR-Cas system allows generation of targeted mutations in mice, even in laboratories with limited or no access to the complex, time-consuming standard technology using mouse embryonic stem cells. Here we summarize the distinct applications of CRISPR-Cas technology for obtaining a variety of genetic modifications in different model organisms, underlining their advantages and limitations relative to other genome editing nucleases. We will guide the reader through the many publications that have seen the light in the first year of CRISPR-Cas technology.

The CCCTC-binding factor (CTCF), which anchors DNA loops that organize the genome into structural domains, has a central role in gene control by facilitating or constraining interactions between genes and their regulatory elements1,2. In cancer cells, the disruption of CTCF binding at specific loci by somatic mutation3,4 or DNA hypermethylation5 results in the loss of loop anchors and consequent activation of oncogenes. By contrast, the germ-cell-specific paralogue of CTCF, BORIS (brother of the regulator of imprinted sites, also known as CTCFL)6, is overexpressed in several cancers7-9, but its contributions to the malignant phenotype remain unclear. Here we show that aberrant upregulation of BORIS promotes chromatin interactions in ALK-mutated, MYCN-amplified neuroblastoma10 cells that develop resistance to ALK inhibition. These cells are reprogrammed to a distinct phenotypic state during the acquisition of resistance, a process defined by the initial loss of MYCN expression followed by subsequent overexpression of BORIS and a concomitant switch in cellular dependence from MYCN to BORIS. The resultant BORIS-regulated alterations in chromatin looping lead to the formation of super-enhancers that drive the ectopic expression of a subset of proneural transcription factors that ultimately define the resistance phenotype. These results identify a previously unrecognized role of BORIS-to promote regulatory chromatin interactions that support specific cancer phenotypes.

Solid tumours are infiltrated by effector T cells with the potential to control or reject them, as well as by regulatory T (Treg) cells that restrict the function of effector T cells and thereby promote tumour growth1. The anti-tumour activity of effector T cells can be therapeutically unleashed, and is now being exploited for the treatment of some forms of human cancer. However, weak tumour-associated inflammatory responses and the immune-suppressive function of Treg cells remain major hurdles to broader effectiveness of tumour immunotherapy2. Here we show that, after disruption of the CARMA1-BCL10-MALT1 (CBM) signalosome complex, most tumour-infiltrating Treg cells produce IFNγ, resulting in stunted tumour growth. Notably, genetic deletion of both or even just one allele of CARMA1 (also known as Card11) in only a fraction of Treg cells-which avoided systemic autoimmunity-was sufficient to produce this anti-tumour effect, showing that it is not the mere loss of suppressive function but the gain of effector activity by Treg cells that initiates tumour control. The production of IFNγ by Treg cells was accompanied by activation of macrophages and upregulation of class I molecules of the major histocompatibility complex on tumour cells. However, tumour cells also upregulated the expression of PD-L1, which indicates activation of adaptive immune resistance3. Consequently, blockade of PD-1 together with CARMA1 deletion caused rejection of tumours that otherwise do not respond to anti-PD-1 monotherapy. This effect was reproduced by pharmacological inhibition of the CBM protein MALT1. Our results demonstrate that partial disruption of the CBM complex and induction of IFNγ secretion in the preferentially self-reactive Treg cell pool does not cause systemic autoimmunity but is sufficient to prime the tumour environment for successful immune checkpoint therapy.

Conditional mutagenesis using Cre recombinase expressed from tissue specific promoters facilitates analyses of gene function and cell lineage tracing. Here, we describe two novel dual-promoter-driven conditional mutagenesis systems designed for greater accuracy and optimal efficiency of recombination. Co-Driver employs a recombinase cascade of Dre and Dre-respondent Cre, which processes loxP-flanked alleles only when both recombinases are expressed in a predetermined temporal sequence. This unique property makes Co-Driver ideal for sequential lineage tracing studies aimed at unraveling the relationships between cellular precursors and mature cell types. Co-InCre was designed for highly efficient intersectional conditional transgenesis. It relies on highly active trans-splicing inteins and promoters with simultaneous transcriptional activity to reconstitute Cre recombinase from two inactive precursor fragments. By generating native Cre, Co-InCre attains recombination rates that exceed all other binary SSR systems evaluated in this study. Both Co-Driver and Co-InCre significantly extend the utility of existing Cre-responsive alleles.

Developmental silencing of fetal globins serves as both a paradigm of spatiotemporal gene regulation and an opportunity for therapeutic intervention of β-hemoglobinopathy. The nucleosome remodeling and deacetylase (NuRD) chromatin complex participates in γ-globin repression. We used pooled CRISPR screening to disrupt NuRD protein coding sequences comprehensively in human adult erythroid precursors. Essential for fetal hemoglobin (HbF) control is a non-redundant subcomplex of NuRD protein family paralogs, whose composition we corroborated by affinity chromatography and proximity labeling mass spectrometry proteomics. Mapping top functional guide RNAs identified key protein interfaces where in-frame alleles resulted in loss-of-function due to destabilization or altered function of subunits. We ascertained mutations of CHD4 that dissociate its requirement for cell fitness from HbF repression in both primary human erythroid precursors and transgenic mice. Finally we demonstrated that sequestering CHD4 from NuRD phenocopied these mutations. These results indicate a generalizable approach to discover protein complex features amenable to rational biochemical targeting.

Approximately, 30% of embryonic stem cells (ESCs) die after exiting self-renewal, but regulators of this process are not well known. Yap1 is a Hippo pathway transcriptional effector that plays numerous roles in development and cancer. However, its functions in ESC differentiation remain poorly characterized. We first reveal that ESCs lacking Yap1 experience massive cell death upon the exit from self-renewal. We subsequently show that Yap1 contextually protects differentiating, but not self-renewing, ESC from hyperactivation of the apoptotic cascade. Mechanistically, Yap1 strongly activates anti-apoptotic genes via cis-regulatory elements while mildly suppressing pro-apoptotic genes, which moderates the level of mitochondrial priming that occurs during differentiation. Individually modulating the expression of single apoptosis-related genes targeted by Yap1 is sufficient to augment or hinder survival during differentiation. Our demonstration of the context-dependent pro-survival functions of Yap1 during ESC differentiation contributes to our understanding of the balance between survival and death during cell fate changes.

Control of gene expression is dictated by cell-type specific regulatory sequences that physically organize the structure of chromatin, including promoters, enhancers and insulators. While promoters and enhancers convey cell-type specific activating signals, insulators prevent the cross-talk of regulatory elements within adjacent loci and safeguard the specificity of action of promoters and enhancers towards their targets in a tissue specific manner. Using the mouse tyrosinase (Tyr) locus as an experimental model, a gene whose mutations are associated with albinism, we described the chromatin structure in cells at two distinct transcriptional states. Guided by chromatin structure, through the use of Chromosome Conformation Capture (3C), we identified sequences at the 5' and 3' boundaries of this mammalian gene that function as enhancers and insulators. By CRISPR/Cas9-mediated chromosomal deletion, we dissected the functions of these two regulatory elements in vivo in the mouse, at the endogenous chromosomal context, and proved their mechanistic role as genomic insulators, shielding the Tyr locus from the expression patterns of adjacent genes.

How overall principles of cell-type-specific gene regulation (the "logic") may change during ontogeny is largely unexplored. We compared transcriptomic, epigenomic, and three-dimensional (3D) genomic profiles in embryonic (EryP) and adult (EryD) erythroblasts. Despite reduced chromatin accessibility compared to EryP, distal chromatin of EryD is enriched in H3K27ac, Gata1, and Myb occupancy. EryP-/EryD-shared enhancers are highly correlated with red blood cell identity genes, whereas cell-type-specific regulation employs different cis elements in EryP and EryD cells. In contrast to EryP-specific genes, which exhibit promoter-centric regulation through Gata1, EryD-specific genes rely more on distal enhancers for regulation involving Myb-mediated enhancer activation. Gata1 HiChIP demonstrated an overall increased enhancer-promoter interactions at EryD-specific genes, whereas genome editing in selected loci confirmed distal enhancers are required for gene expression in EryD but not in EryP. Applying a metric for enhancer dependence of transcription, we observed a progressive reliance on cell-specific enhancers with increasing ontogenetic age among diverse tissues of mouse and human origin. Our findings highlight fundamental and conserved differences at distinct developmental stages, characterized by simpler promoter-centric regulation of cell-type-specific genes in embryonic cells and increased combinatorial enhancer-driven control in adult cells.

CRISPR/Cas9 screening approaches are powerful tool for identifying in vivo cancer dependencies. Hematopoietic malignancies are genetically complex disorders in which the sequential acquisition of somatic mutations generates clonal diversity. Over time, additional cooperating mutations may drive disease progression. Using an in vivo pooled gene editing screen of epigenetic factors in primary murine hematopoietic stem and progenitor cells (HSPCs), we sought to uncover unrecognized genes that contribute to leukemia progression. We, first, modeled myeloid leukemia in mice by functionally abrogating both Tet2 and Tet3 in HSPCs, followed by transplantation. We, then, performed pooled CRISPR/Cas9 editing of genes encoding epigenetic factors and identified Pbrm1/Baf180, a subunit of the polybromo BRG1/BRM-associated factor SWItch/Sucrose Non-Fermenting chromatin-remodeling complex, as a negative driver of disease progression. We found that Pbrm1 loss promoted leukemogenesis with a significantly shortened latency. Pbrm1-deficient leukemia cells were less immunogenic and were characterized by attenuated interferon signaling and reduced major histocompatibility complex class II (MHC II) expression. We explored the potential relevance to human leukemia by assessing the involvement of PBRM1 in the control of interferon pathway components and found that PBRM1 binds to the promoters of a subset of these genes, most notably IRF1, which in turn regulates MHC II expression. Our findings revealed a novel role for Pbrm1 in leukemia progression. More generally, CRISPR/Cas9 screening coupled with phenotypic readouts in vivo has helped identify a pathway by which transcriptional control of interferon signaling influences leukemia cell interactions with the immune system.