Single-cell epigenome analysis identifies molecular events controlling direct conversion of human fibroblasts to pancreatic ductal-like cells.

Cell fate can be reprogrammed by ectopic expression of lineage-specific transcription factors (TFs). However, the exact cell state transitions during transdifferentiation are still poorly understood. Here, we have generated pancreatic exocrine cells of ductal epithelial identity from human fibroblasts using a set of six TFs. We mapped the molecular determinants of lineage dynamics using a factor-indexing method based on single-nuclei multiome sequencing (FI-snMultiome-seq) that enables dissecting the role of each individual TF and pool of TFs in cell fate conversion. We show that transition from mesenchymal fibroblast identity to epithelial pancreatic exocrine fate involves two deterministic steps: an endodermal progenitor state defined by activation of HHEX with FOXA2 and SOX17 and a temporal GATA4 activation essential for the maintenance of pancreatic cell fate program. Collectively, our data suggest that transdifferentiation-although being considered a direct cell fate conversion method-occurs through transient progenitor states orchestrated by stepwise activation of distinct TFs.

Transposable elements as tissue-specific enhancers in cancers of endodermal lineage.

Transposable elements (TE) are repetitive genomic elements that harbor binding sites for human transcription factors (TF). A regulatory role for TEs has been suggested in embryonal development and diseases such as cancer but systematic investigation of their functions has been limited by their widespread silencing in the genome. Here, we utilize unbiased massively parallel reporter assay data using a whole human genome library to identify TEs with functional enhancer activity in two human cancer types of endodermal lineage, colorectal and liver cancers. We show that the identified TE enhancers are characterized by genomic features associated with active enhancers, such as epigenetic marks and TF binding. Importantly, we identify distinct TE subfamilies that function as tissue-specific enhancers, namely MER11- and LTR12-elements in colon and liver cancers, respectively. These elements are bound by distinct TFs in each cell type, and they have predicted associations to differentially expressed genes. In conclusion, these data demonstrate how different cancer types can utilize distinct TEs as tissue-specific enhancers, paving the way for comprehensive understanding of the role of TEs as bona fide enhancers in the cancer genomes.

Comprehensive characterization of the embryonic factor LEUTX.

The paired-like homeobox transcription factor LEUTX is expressed in human preimplantation embryos between the 4- and 8-cell stages, and then silenced in somatic tissues. To characterize the function of LEUTX, we performed a multiomic characterization of LEUTX using two proteomics methods and three genome-wide sequencing approaches. Our results show that LEUTX stably interacts with the EP300 and CBP histone acetyltransferases through its 9 amino acid transactivation domain (9aaTAD), as mutation of this domain abolishes the interactions. LEUTX targets genomic cis-regulatory sequences that overlap with repetitive elements, and through these elements it is suggested to regulate the expression of its downstream genes. We find LEUTX to be a transcriptional activator, upregulating several genes linked to preimplantation development as well as 8-cell-like markers, such as DPPA3 and ZNF280A. Our results support a role for LEUTX in preimplantation development as an enhancer binding protein and as a potent transcriptional activator.

A competitive precision CRISPR method to identify the fitness effects of transcription factor binding sites.

Here we describe a competitive genome editing method that measures the effect of mutations on molecular functions, based on precision CRISPR editing using template libraries with either the original or altered sequence, and a sequence tag, enabling direct comparison between original and mutated cells. Using the example of the MYC oncogene, we identify important transcriptional targets and show that E-box mutations at MYC target gene promoters reduce cellular fitness.

Sequence determinants of human gene regulatory elements.

DNA can determine where and when genes are expressed, but the full set of sequence determinants that control gene expression is unknown. Here, we measured the transcriptional activity of DNA sequences that represent an ~100 times larger sequence space than the human genome using massively parallel reporter assays (MPRAs). Machine learning models revealed that transcription factors (TFs) generally act in an additive manner with weak grammar and that most enhancers increase expression from a promoter by a mechanism that does not appear to involve specific TF-TF interactions. The enhancers themselves can be classified into three types: classical, closed chromatin and chromatin dependent. We also show that few TFs are strongly active in a cell, with most activities being similar between cell types. Individual TFs can have multiple gene regulatory activities, including chromatin opening and enhancing, promoting and determining transcription start site (TSS) activity, consistent with the view that the TF binding motif is the key atomic unit of gene expression.

Compressive stress-mediated p38 activation required for ERα + phenotype in breast cancer.

Breast cancer is now globally the most frequent cancer and leading cause of women's death. Two thirds of breast cancers express the luminal estrogen receptor-positive (ERα + ) phenotype that is initially responsive to antihormonal therapies, but drug resistance emerges. A major barrier to the understanding of the ERα-pathway biology and therapeutic discoveries is the restricted repertoire of luminal ERα + breast cancer models. The ERα + phenotype is not stable in cultured cells for reasons not fully understood. We examine 400 patient-derived breast epithelial and breast cancer explant cultures (PDECs) grown in various three-dimensional matrix scaffolds, finding that ERα is primarily regulated by the matrix stiffness. Matrix stiffness upregulates the ERα signaling via stress-mediated p38 activation and H3K27me3-mediated epigenetic regulation. The finding that the matrix stiffness is a central cue to the ERα phenotype reveals a mechanobiological component in breast tissue hormonal signaling and enables the development of novel therapeutic interventions. Subject terms: ER-positive (ER + ), breast cancer, ex vivo model, preclinical model, PDEC, stiffness, p38 SAPK.

Deficient H2A.Z deposition is associated with genesis of uterine leiomyoma.

One in four women suffers from uterine leiomyomas (ULs)-benign tumours of the uterine wall, also known as uterine fibroids-at some point in premenopausal life. ULs can cause excessive bleeding, pain and infertility1, and are a common cause of hysterectomy2. They emerge through at least three distinct genetic drivers: mutations in MED12 or FH, or genomic rearrangement of HMGA23. Here we created genome-wide datasets, using DNA, RNA, assay for transposase-accessible chromatin (ATAC), chromatin immunoprecipitation (ChIP) and HiC chromatin immunoprecipitation (HiChIP) sequencing of primary tissues to profoundly understand the genesis of UL. We identified somatic mutations in genes encoding six members of the SRCAP histone-loading complex4, and found that germline mutations in the SRCAP members YEATS4 and ZNHIT1 predispose women to UL. Tumours bearing these mutations showed defective deposition of the histone variant H2A.Z. In ULs, H2A.Z occupancy correlated positively with chromatin accessibility and gene expression, and negatively with DNA methylation, but these correlations were weak in tumours bearing SRCAP complex mutations. In these tumours, open chromatin emerged at transcription start sites where H2A.Z was lost, which was associated with upregulation of genes. Furthermore, YEATS4 defects were associated with abnormal upregulation of bivalent embryonic stem cell genes, as previously shown in mice5. Our work describes a potential mechanism of tumorigenesis-epigenetic instability caused by deficient H2A.Z deposition-and suggests that ULs arise through an aberrant differentiation program driven by deranged chromatin, emanating from a small number of mutually exclusive driver mutations.

Human cell transformation by combined lineage conversion and oncogene expression.

Cancer is the most complex genetic disease known, with mutations implicated in more than 250 genes. However, it is still elusive which specific mutations found in human patients lead to tumorigenesis. Here we show that a combination of oncogenes that is characteristic of liver cancer (CTNNB1, TERT, MYC) induces senescence in human fibroblasts and primary hepatocytes. However, reprogramming fibroblasts to a liver progenitor fate, induced hepatocytes (iHeps), makes them sensitive to transformation by the same oncogenes. The transformed iHeps are highly proliferative, tumorigenic in nude mice, and bear gene expression signatures of liver cancer. These results show that tumorigenesis is triggered by a combination of three elements: the set of driver mutations, the cellular lineage, and the state of differentiation of the cells along the lineage. Our results provide direct support for the role of cell identity as a key determinant in transformation and establish a paradigm for studying the dynamic role of oncogenic drivers in human tumorigenesis.

Gain-of-function CEBPE mutation causes noncanonical autoinflammatory inflammasomopathy.

BACKGROUND: CCAAT enhancer-binding protein epsilon (C/EBPε) is a transcription factor involved in late myeloid lineage differentiation and cellular function. The only previously known disorder linked to C/EBPε is autosomal recessive neutrophil-specific granule deficiency leading to severely impaired neutrophil function and early mortality. OBJECTIVE: The aim of this study was to molecularly characterize the effects of C/EBPε transcription factor Arg219His mutation identified in a Finnish family with previously genetically uncharacterized autoinflammatory and immunodeficiency syndrome. METHODS: Genetic analysis, proteomics, genome-wide transcriptional profiling by means of RNA-sequencing, chromatin immunoprecipitation (ChIP) sequencing, and assessment of the inflammasome function of primary macrophages were performed. RESULTS: Studies revealed a novel mechanism of genome-wide gain-of-function that dysregulated transcription of 464 genes. Mechanisms involved dysregulated noncanonical inflammasome activation caused by decreased association with transcriptional repressors, leading to increased chromatin occupancy and considerable changes in transcriptional activity, including increased expression of NLR family, pyrin domain-containing 3 protein (NLRP3) and constitutively expressed caspase-5 in macrophages. CONCLUSION: We describe a novel autoinflammatory disease with defective neutrophil function caused by a homozygous Arg219His mutation in the transcription factor C/EBPε. Mutated C/EBPε acts as a regulator of both the inflammasome and interferome, and the Arg219His mutation causes the first human monogenic neomorphic and noncanonical inflammasomopathy/immunodeficiency. The mechanism, including widely dysregulated transcription, is likely not unique for C/EBPε. Similar multiomics approaches should also be used in studying other transcription factor-associated diseases.

The interaction landscape between transcription factors and the nucleosome.

Nucleosomes cover most of the genome and are thought to be displaced by transcription factors in regions that direct gene expression. However, the modes of interaction between transcription factors and nucleosomal DNA remain largely unknown. Here we systematically explore interactions between the nucleosome and 220 transcription factors representing diverse structural families. Consistent with earlier observations, we find that the majority of the studied transcription factors have less access to nucleosomal DNA than to free DNA. The motifs recovered from transcription factors bound to nucleosomal and free DNA are generally similar. However, steric hindrance and scaffolding by the nucleosome result in specific positioning and orientation of the motifs. Many transcription factors preferentially bind close to the end of nucleosomal DNA, or to periodic positions on the solvent-exposed side of the DNA. In addition, several transcription factors usually bind to nucleosomal DNA in a particular orientation. Some transcription factors specifically interact with DNA located at the dyad position at which only one DNA gyre is wound, whereas other transcription factors prefer sites spanning two DNA gyres and bind specifically to each of them. Our work reveals notable differences in the binding of transcription factors to free and nucleosomal DNA, and uncovers a diverse interaction landscape between transcription factors and the nucleosome.

Contribution of allelic imbalance to colorectal cancer.

Point mutations in cancer have been extensively studied but chromosomal gains and losses have been more challenging to interpret due to their unspecific nature. Here we examine high-resolution allelic imbalance (AI) landscape in 1699 colorectal cancers, 256 of which have been whole-genome sequenced (WGSed). The imbalances pinpoint 38 genes as plausible AI targets based on previous knowledge. Unbiased CRISPR-Cas9 knockout and activation screens identified in total 79 genes within AI peaks regulating cell growth. Genetic and functional data implicate loss of TP53 as a sufficient driver of AI. The WGS highlights an influence of copy number aberrations on the rate of detected somatic point mutations. Importantly, the data reveal several associations between AI target genes, suggesting a role for a network of lineage-determining transcription factors in colorectal tumorigenesis. Overall, the results unravel the contribution of AI in colorectal cancer and provide a plausible explanation why so few genes are commonly affected by point mutations in cancers.

A protein activity assay to measure global transcription factor activity reveals determinants of chromatin accessibility.

No existing method to characterize transcription factor (TF) binding to DNA allows genome-wide measurement of all TF-binding activity in cells. Here we present a massively parallel protein activity assay, active TF identification (ATI), that measures the DNA-binding activity of all TFs in cell or tissue extracts. ATI is based on electrophoretic separation of protein-bound DNA sequences from a highly complex DNA library and subsequent mass-spectrometric identification of the DNA-bound proteins. We applied ATI to four mouse tissues and mouse embryonic stem cells and found that, in a given tissue or cell type, a small set of TFs, which bound to only ∼10 distinct motifs, displayed strong DNA-binding activity. Some of these TFs were found in all cell types, whereas others were specific TFs known to determine cell fate in the analyzed tissue or cell type. We also show that a small number of TFs determined the accessible chromatin landscape of a cell, suggesting that gene regulatory logic may be simpler than previously appreciated.

Characterization of an androgen-responsive, ornithine decarboxylase-related protein in mouse kidney.

We have investigated and characterized a novel ornithine decarboxylase (ODC) related protein (ODCrp) also annotated as gm853. ODCrp shows 41% amino acid sequence identity with ODC and 38% with ODC antizyme inhibitor 1 (AZIN1). The Odcrp gene is selectively expressed in the epithelium of proximal tubuli of mouse kidney with higher expression in males than in females. Like Odc in mouse kidney, Odcrp is also androgen responsive with androgen receptor (AR)-binding loci within its regulatory region. ODCrp forms homodimers but does not heterodimerize with ODC. Although ODCrp contains 20 amino acid residues known to be necessary for the catalytic activity of ODC, no decarboxylase activity could be found with ornithine, lysine or arginine as substrates. ODCrp does not function as an AZIN, as it neither binds ODC antizyme 1 (OAZ1) nor prevents OAZ-mediated inactivation and degradation of ODC. ODCrp itself is degraded via ubiquination and mutation of Cys363 (corresponding to Cys360 of ODC) appears to destabilize the protein. Evidence for a function of ODCrp was found in ODC assays on lysates from transfected Cos-7 cells where ODCrp repressed the activity of endogenous ODC while Cys363Ala mutated ODCrp increased the enzymatic activity of endogenous ODC.

Impact of cytosine methylation on DNA binding specificities of human transcription factors.

The majority of CpG dinucleotides in the human genome are methylated at cytosine bases. However, active gene regulatory elements are generally hypomethylated relative to their flanking regions, and the binding of some transcription factors (TFs) is diminished by methylation of their target sequences. By analysis of 542 human TFs with methylation-sensitive SELEX (systematic evolution of ligands by exponential enrichment), we found that there are also many TFs that prefer CpG-methylated sequences. Most of these are in the extended homeodomain family. Structural analysis showed that homeodomain specificity for methylcytosine depends on direct hydrophobic interactions with the methylcytosine 5-methyl group. This study provides a systematic examination of the effect of an epigenetic DNA modification on human TF binding specificity and reveals that many developmentally important proteins display preference for mCpG-containing sequences.

Damaging heterozygous mutations in NFKB1 lead to diverse immunologic phenotypes.

BACKGROUND: The nuclear factor κ light-chain enhancer of activated B cells (NF-κB) signaling pathway is a key regulator of immune responses. Accordingly, mutations in several NF-κB pathway genes cause immunodeficiency. OBJECTIVE: We sought to identify the cause of disease in 3 unrelated Finnish kindreds with variable symptoms of immunodeficiency and autoinflammation. METHODS: We applied genetic linkage analysis and next-generation sequencing and functional analyses of NFKB1 and its mutated alleles. RESULTS: In all affected subjects we detected novel heterozygous variants in NFKB1, encoding for p50/p105. Symptoms in variant carriers differed depending on the mutation. Patients harboring a p.I553M variant presented with antibody deficiency, infection susceptibility, and multiorgan autoimmunity. Patients with a p.H67R substitution had antibody deficiency and experienced autoinflammatory episodes, including aphthae, gastrointestinal disease, febrile attacks, and small-vessel vasculitis characteristic of Behçet disease. Patients with a p.R157X stop-gain experienced hyperinflammatory responses to surgery and showed enhanced inflammasome activation. In functional analyses the p.R157X variant caused proteasome-dependent degradation of both the truncated and wild-type proteins, leading to a dramatic loss of p50/p105. The p.H67R variant reduced nuclear entry of p50 and showed decreased transcriptional activity in luciferase reporter assays. The p.I553M mutation in turn showed no change in p50 function but exhibited reduced p105 phosphorylation and stability. Affinity purification mass spectrometry also demonstrated that both missense variants led to altered protein-protein interactions. CONCLUSION: Our findings broaden the scope of phenotypes caused by mutations in NFKB1 and suggest that a subset of autoinflammatory diseases, such as Behçet disease, can be caused by rare monogenic variants in genes of the NF-κB pathway.

PeakXus: comprehensive transcription factor binding site discovery from ChIP-Nexus and ChIP-Exo experiments.

MOTIVATION: Transcription factor (TF) binding can be studied accurately in vivo with ChIP-exo and ChIP-Nexus experiments. Only fraction of TF binding mechanisms are yet fully understood and accurate knowledge of binding locations and patterns of TFs is key to understanding binding that is not explained by simple positional weight matrix models. ChIP-exo/Nexus experiments can also offer insight on the effect of single nucleotide polymorphism (SNP) at TF binding sites on expression of the target genes. This is an important mechanism of action for disease-causing SNPs at non-coding genomic regions. RESULTS: We describe a peak caller PeakXus that is specifically designed to leverage the increased resolution of ChIP-exo/Nexus and developed with the aim of making as few assumptions of the data as possible to allow discoveries of novel binding patterns. We apply PeakXus to ChIP-Nexus and ChIP-exo experiments performed both in Homo sapiens and in Drosophila melanogaster cell lines. We show that PeakXus consistently finds more peaks overlapping with a TF-specific recognition sequence than published methods. As an application example we demonstrate how PeakXus can be coupled with unique molecular identifiers (UMIs) to measure the effect of a SNP overlapping with a TF binding site on the in vivo binding of the TF. AVAILABILITY AND IMPLEMENTATION: Source code of PeakXus is available at https://github.com/hartonen/PeakXus CONTACT: tuomo.hartonen@helsinki.fi or jussi.taipale@ki.se.

Androgen receptor- and PIAS1-regulated gene programs in molecular apocrine breast cancer cells.

We have analyzed androgen receptor (AR) chromatin binding sites (ARBs) and androgen-regulated transcriptome in estrogen receptor negative molecular apocrine breast cancer cells. These analyses revealed that 42% of ARBs and 39% androgen-regulated transcripts in MDA-MB453 cells have counterparts in VCaP prostate cancer cells. Pathway analyses showed a similar enrichment of molecular and cellular functions among AR targets in both breast and prostate cancer cells, with cellular growth and proliferation being among the most enriched functions. Silencing of the coregulator SUMO ligase PIAS1 in MDA-MB453 cells influenced AR function in a target-selective fashion. An anti-apoptotic effect of the silencing suggests involvement of the PIAS1 in the regulation of cell death and survival pathways. In sum, apocrine breast cancer and prostate cancer cells share a core AR cistrome and target gene signature linked to cancer cell growth, and PIAS1 plays a similar coregulatory role for AR in both cancer cell types.

Determinants of Receptor- and Tissue-Specific Actions in Androgen Signaling.

The physiological androgens testosterone and 5α-dihydrotestosterone regulate the development and maintenance of primary and secondary male sexual characteristics through binding to the androgen receptor (AR), a ligand-dependent transcription factor. In addition, a number of nonreproductive tissues of both genders are subject to androgen regulation. AR is also a central target in the treatment of prostate cancer. A large number of studies over the last decade have characterized many regulatory aspects of the AR pathway, such as androgen-dependent transcription programs, AR cistromes, and coregulatory proteins, mostly in cultured cells of prostate cancer origin. Moreover, recent work has revealed the presence of pioneer/licensing factors and chromatin modifications that are important to guide receptor recruitment onto appropriate chromatin loci in cell lines and in tissues under physiological conditions. Despite these advances, current knowledge related to the mechanisms responsible for receptor- and tissue-specific actions of androgens is still relatively limited. Here, we review topics that pertain to these specificity issues at different levels, both in cultured cells and tissues in vivo, with a particular emphasis on the nature of the steroid, the response element sequence, the AR cistromes, pioneer/licensing factors, and coregulatory proteins. We conclude that liganded AR and its DNA-response elements are required but are not sufficient for establishment of tissue-specific transcription programs in vivo, and that AR-selective actions over other steroid receptors rely on relaxed rather than increased stringency of cis-elements on chromatin.

Identification of several potential chromatin binding sites of HOXB7 and its downstream target genes in breast cancer.

HOXB7 encodes a transcription factor that is overexpressed in a number of cancers and encompasses many oncogenic functions. Previous results have shown it to promote cell proliferation, angiogenesis, epithelial-mesenchymal transition, DNA repair and cell survival. Because of its role in many cancers and tumorigenic processes, HOXB7 has been suggested to be a potential drug target. However, HOXB7 binding sites on chromatin and its targets are poorly known. The aim of our study was to identify HOXB7 binding sites on breast cancer cell chromatin and to delineate direct target genes located nearby these binding sites. We found 1,504 HOXB7 chromatin binding sites in BT-474 breast cancer cell line that overexpresses HOXB7. Seventeen selected binding sites were validated by ChIP-qPCR in several breast cancer cell lines. Furthermore, we analyzed expression of a large number of genes located nearby HOXB7 binding sites and found several new direct targets, such as CTNND2 and SCGB1D2. Identification of HOXB7 chromatin binding sites and target genes is essential to understand better the role of HOXB7 in breast cancer and mechanisms by which it regulates tumorigenic processes.

SUMO ligase PIAS1 functions as a target gene selective androgen receptor coregulator on prostate cancer cell chromatin.

Androgen receptor (AR) is a ligand-activated transcription factor that plays a central role in the development and growth of prostate carcinoma. PIAS1 is an AR- and SUMO-interacting protein and a putative transcriptional coregulator overexpressed in prostate cancer. To study the importance of PIAS1 for the androgen-regulated transcriptome of VCaP prostate cancer cells, we silenced its expression by RNAi. Transcriptome analyses revealed that a subset of the AR-regulated genes is significantly influenced, either activated or repressed, by PIAS1 depletion. Interestingly, PIAS1 depletion also exposed a new set of genes to androgen regulation, suggesting that PIAS1 can mask distinct genomic loci from AR access. In keeping with gene expression data, silencing of PIAS1 attenuated VCaP cell proliferation. ChIP-seq analyses showed that PIAS1 interacts with AR at chromatin sites harboring also SUMO2/3 and surrounded by H3K4me2; androgen exposure increased the number of PIAS1-occupying sites, resulting in nearly complete overlap with AR chromatin binding events. PIAS1 interacted also with the pioneer factor FOXA1. Of note, PIAS1 depletion affected AR chromatin occupancy at binding sites enriched for HOXD13 and GATA motifs. Taken together, PIAS1 is a genuine chromatin-bound AR coregulator that functions in a target gene selective fashion to regulate prostate cancer cell growth.