Cerebro: interactive visualization of scRNA-seq data.

Despite the growing availability of sophisticated bioinformatic methods for the analysis of single-cell RNA-seq data, few tools exist that allow biologists without extensive bioinformatic expertise to directly visualize and interact with their own data and results. Here, we present Cerebro (cell report browser), a Shiny- and Electron-based standalone desktop application for macOS and Windows which allows investigation and inspection of pre-processed single-cell transcriptomics data without requiring bioinformatic experience of the user. Through an interactive and intuitive graphical interface, users can (i) explore similarities and heterogeneity between samples and cell clusters in two-dimensional or three-dimensional projections such as t-SNE or UMAP, (ii) display the expression level of single genes or gene sets of interest, (iii) browse tables of most expressed genes and marker genes for each sample and cluster and (iv) display trajectories calculated with Monocle 2. We provide three examples prepared from publicly available datasets to show how Cerebro can be used and which are its capabilities. Through a focus on flexibility and direct access to data and results, we think Cerebro offers a collaborative framework for bioinformaticians and experimental biologists that facilitates effective interaction to shorten the gap between analysis and interpretation of the data. AVAILABILITY AND IMPLEMENTATION: The Cerebro application, additional documentation, and example datasets are available at https://github.com/romanhaa/Cerebro. Similarly, the cerebroApp R package is available at https://github.com/romanhaa/cerebroApp. All components are released under the MIT License. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A knowledge-based framework for the discovery of cancer-predisposing variants using large-scale sequencing breast cancer data.

BACKGROUND: The landscape of cancer-predisposing genes has been extensively investigated in the last 30 years with various methodologies ranging from candidate gene to genome-wide association studies. However, sequencing data are still poorly exploited in cancer predisposition studies due to the lack of statistical power when comparing millions of variants at once. METHOD: To overcome these power limitations, we propose a knowledge-based framework founded on the characteristics of known cancer-predisposing variants and genes. Under our framework, we took advantage of a combination of previously generated datasets of sequencing experiments to identify novel breast cancer-predisposing variants, comparing the normal genomes of 673 breast cancer patients of European origin against 27,173 controls matched by ethnicity. RESULTS: We detected several expected variants on known breast cancer-predisposing genes, like BRCA1 and BRCA2, and 11 variants on genes associated with other cancer types, like RET and AKT1. Furthermore, we detected 183 variants that overlap with somatic mutations in cancer and 41 variants associated with 38 possible loss-of-function genes, including PIK3CB and KMT2C. Finally, we found a set of 19 variants that are potentially pathogenic, negatively correlate with age at onset, and have never been associated with breast cancer. CONCLUSIONS: In this study, we demonstrate the usefulness of a genomic-driven approach nested in a classic case-control study to prioritize cancer-predisposing variants. In addition, we provide a resource containing variants that may affect susceptibility to breast cancer.

The hidden genomic landscape of acute myeloid leukemia: subclonal structure revealed by undetected mutations.

The analyses carried out using 2 different bioinformatics pipelines (SomaticSniper and MuTect) on the same set of genomic data from 133 acute myeloid leukemia (AML) patients, sequenced inside the Cancer Genome Atlas project, gave discrepant results. We subsequently tested these 2 variant-calling pipelines on 20 leukemia samples from our series (19 primary AMLs and 1 secondary AML). By validating many of the predicted somatic variants (variant allele frequencies ranging from 100% to 5%), we observed significantly different calling efficiencies. In particular, despite relatively high specificity, sensitivity was poor in both pipelines resulting in a high rate of false negatives. Our findings raise the possibility that landscapes of AML genomes might be more complex than previously reported and characterized by the presence of hundreds of genes mutated at low variant allele frequency, suggesting that the application of genome sequencing to the clinic requires a careful and critical evaluation. We think that improvements in technology and workflow standardization, through the generation of clear experimental and bioinformatics guidelines, are fundamental to translate the use of next-generation sequencing from research to the clinic and to transform genomic information into better diagnosis and outcomes for the patient.

Genome-wide mapping of human DNA-replication origins: levels of transcription at ORC1 sites regulate origin selection and replication timing.

We report the genome-wide mapping of ORC1 binding sites in mammals, by chromatin immunoprecipitation and parallel sequencing (ChIP-seq). ORC1 binding sites in HeLa cells were validated as active DNA replication origins (ORIs) using Repli-seq, a method that allows identification of ORI-containing regions by parallel sequencing of temporally ordered replicating DNA. ORC1 sites were universally associated with transcription start sites (TSSs) of coding or noncoding RNAs (ncRNAs). Transcription levels at the ORC1 sites directly correlated with replication timing, suggesting the existence of two classes of ORIs: those associated with moderate/high transcription levels (≥1 RNA copy/cell), firing in early S and mapping to the TSSs of coding RNAs; and those associated with low transcription levels (<1 RNA copy/cell), firing throughout the entire S and mapping to TSSs of ncRNAs. These findings are compatible with a scenario whereby TSS expression levels influence the efficiency of ORC1 recruitment at G(1) and the probability of firing during S.

Myc-binding-site recognition in the human genome is determined by chromatin context.

Large-scale chromatin immunoprecipitation (ChIP) studies have been effective in unravelling the distribution of DNA-binding transcription factors along eukaryotic genomes, but specificity determinants remain elusive. Gene-regulatory regions display distinct histone variants and modifications (or marks). An attractive hypothesis is that these marks modulate protein recognition, but whether or not this applies to transcription factors remains unknown. Based on large-scale datasets and quantitative ChIP, we dissect the correlations between 35 histone marks and genomic binding by the transcription factor Myc. Our data reveal a relatively simple combinatorial organization of histone marks in human cells, with a few main groups of marks clustering on distinct promoter populations. A stretch of chromatin bearing high H3 K4/K79 methylation and H3 acetylation (or 'euchromatic island'), which is generally associated with a pre-engaged basal transcription machinery, is a strict pre-requisite for recognition of any target site by Myc (whether the consensus CACGTG or an alternative sequence). These data imply that tethering of a transcription factor to restricted chromatin domains is rate-limiting for sequence-specific DNA binding in vivo.

A Rare Subset of Primary Tumor Cells with Concomitant Hyperactivation of Extracellular Matrix Remodeling and dsRNA-IFN1 Signaling Metastasizes in Breast Cancer.

Metastatic breast cancer has a poor prognosis and is largely considered incurable. A better understanding of the molecular determinants of breast cancer metastasis could facilitate development of improved prevention and treatment strategies. We used lentiviral barcoding coupled to single-cell RNA sequencing to trace clonal and transcriptional evolution during breast cancer metastasis and showed that metastases derive from rare prometastatic clones that are underrepresented in primary tumors. Both low clonal fitness and high metastatic potential were independent of clonal origin. Differential expression and classification analyses revealed that the prometastatic phenotype was acquired by rare cells characterized by the concomitant hyperactivation of extracellular matrix remodeling and dsRNA-IFN signaling pathways. Notably, genetic silencing of key genes in these pathways (KCNQ1OT1 or IFI6, respectively) significantly impaired migration in vitro and metastasis in vivo, with marginal effects on cell proliferation and tumor growth. Gene expression signatures derived from the identified prometastatic genes predict metastatic progression in patients with breast cancer, independently of known prognostic factors. This study elucidates previously unknown mechanisms of breast cancer metastasis and provides prognostic predictors and therapeutic targets for metastasis prevention. SIGNIFICANCE: Transcriptional lineage tracing coupled with single-cell transcriptomics defined the transcriptional programs underlying metastatic progression in breast cancer, identifying prognostic signatures and prevention strategies.

Time makes histone H3 modifications drift in mouse liver.

To detect the epigenetic drift of time passing, we determined the genome-wide distributions of mono- and tri-methylated lysine 4 and acetylated and tri-methylated lysine 27 of histone H3 in the livers of healthy 3, 6 and 12 months old C57BL/6 mice. The comparison of different age profiles of histone H3 marks revealed global redistribution of histone H3 modifications with time, in particular in intergenic regions and near transcription start sites, as well as altered correlation between the profiles of different histone modifications. Moreover, feeding mice with caloric restriction diet, a treatment known to retard aging, reduced the extent of changes occurring during the first year of life in these genomic regions.

Aberrant activation of p53/p66Shc-mInsc axis increases asymmetric divisions and attenuates proliferation of aged mammary stem cells.

Aging is accompanied by the progressive decline in tissue regenerative capacity and functions of resident stem cells (SCs). Underlying mechanisms, however, remain unclear. Here we show that, during chronological aging, self-renewing mitoses of mammary SCs (MaSCs) are preferentially asymmetric and that their progeny divides less frequently, leading to decreased number of MaSCs and reduced regenerative potential. Underlying mechanisms are investigated in the p66Shc-/- mouse, which exhibits several features of delayed aging, including reduced involution of the mammary gland (MG). p66Shc is a mitochondrial redox sensor that activates a specific p53 transcriptional program, in which the aging-associated p44 isoform of p53 plays a pivotal role. We report here that aged p66Shc-/- MaSCs show increased symmetric divisions, increased proliferation and increased regenerative potential, to an extent reminiscent of young wild-type (WT) MaSCs. Mechanistically, we demonstrate that p66Shc, together with p53: (i) accumulates in the aged MG, (ii) sustains expression of the cell polarity determinant mInscuteable and, concomitantly, (iii) down-regulates critical cell cycle genes (e.g.,: Cdk1 and Cyclin A). Accordingly, overexpression of p53/p44 increases asymmetric divisions and decreases proliferation of young WT MaSCs in a p66Shc-dependent manner and overexpression of mInsc restores WT-like levels of asymmetric divisions in aged p66Shc-/- MaSCs. Notably, deletion of p66Shc has negligible effects in young MaSCs and MG development. These results demonstrate that MG aging is due to aberrant activation of p66Shc, which induces p53/p44 signaling, leading to failure of symmetric divisions, decreased proliferation and reduced regenerative potential of MaSCs.

AML1/ETO oncoprotein is directed to AML1 binding regions and co-localizes with AML1 and HEB on its targets.

A reciprocal translocation involving chromosomes 8 and 21 generates the AML1/ETO oncogenic transcription factor that initiates acute myeloid leukemia by recruiting co-repressor complexes to DNA. AML1/ETO interferes with the function of its wild-type counterpart, AML1, by directly targeting AML1 binding sites. However, transcriptional regulation determined by AML1/ETO probably relies on a more complex network, since the fusion protein has been shown to interact with a number of other transcription factors, in particular E-proteins, and may therefore target other sites on DNA. Genome-wide chromatin immunoprecipitation and expression profiling were exploited to identify AML1/ETO-dependent transcriptional regulation. AML1/ETO was found to co-localize with AML1, demonstrating that the fusion protein follows the binding pattern of the wild-type protein but does not function primarily by displacing it. The DNA binding profile of the E-protein HEB was grossly rearranged upon expression of AML1/ETO, and the fusion protein was found to co-localize with both AML1 and HEB on many of its regulated targets. Furthermore, the level of HEB protein was increased in both primary cells and cell lines expressing AML1/ETO. Our results suggest a major role for the functional interaction of AML1/ETO with AML1 and HEB in transcriptional regulation determined by the fusion protein.

XenoCell: classification of cellular barcodes in single cell experiments from xenograft samples.

BACKGROUND: Single-cell sequencing technologies provide unprecedented opportunities to deconvolve the genomic, transcriptomic or epigenomic heterogeneity of complex biological systems. Its application in samples from xenografts of patient-derived biopsies (PDX), however, is limited by the presence of cells originating from both the host and the graft in the analysed samples; in fact, in the bioinformatics workflows it is still a challenge discriminating between host and graft sequence reads obtained in a single-cell experiment. RESULTS: We have developed XenoCell, the first stand-alone pre-processing tool that performs fast and reliable classification of host and graft cellular barcodes from single-cell sequencing experiments. We show its application on a mixed species 50:50 cell line experiment from 10× Genomics platform, and on a publicly available PDX dataset obtained by Drop-Seq. CONCLUSIONS: XenoCell accurately dissects sequence reads from any host and graft combination of species as well as from a broad range of single-cell experiments and platforms. It is open source and available at https://gitlab.com/XenoCell/XenoCell .

Cyclophosphamide and Vinorelbine Activate Stem-Like CD8+ T Cells and Improve Anti-PD-1 Efficacy in Triple-Negative Breast Cancer.

Checkpoint inhibitors (CI) instigate anticancer immunity in many neoplastic diseases, albeit only in a fraction of patients. The clinical success of cyclophosphamide (C)-based haploidentical stem-cell transplants indicates that this drug may re-orchestrate the immune system. Using models of triple-negative breast cancer (TNBC) with different intratumoral immune contexture, we demonstrate that a combinatorial therapy of intermittent C, CI, and vinorelbine activates antigen-presenting cells (APC), and abrogates local and metastatic tumor growth by a T-cell-related effect. Single-cell transcriptome analysis of >50,000 intratumoral immune cells after therapy treatment showed a gene signature suggestive of a change resulting from exposure to a mitogen, ligand, or antigen for which it is specific, as well as APC-to-T-cell adhesion. This transcriptional program also increased intratumoral Tcf1+ stem-like CD8+ T cells and altered the balance between terminally and progenitor-exhausted T cells favoring the latter. Overall, our data support the clinical investigation of this therapy in TNBC. SIGNIFICANCE: A combinatorial therapy in mouse models of breast cancer increases checkpoint inhibition by activating antigen-presenting cells, enhancing intratumoral Tcf1+ stem-like CD8+ T cells, and increasing progenitor exhausted CD8+ T cells.

CARPET: a web-based package for the analysis of ChIP-chip and expression tiling data.

SUMMARY: CARPET (collection of automated routine programs for easy tiling) is a set of Perl, Python and R scripts, integrated on the Galaxy2 web-based platform, for the analysis of ChIP-chip and expression tiling data, both for standard and custom chip designs. CARPET allows rapid experimental data entry, simple quality control, normalization, easy identification and annotation of enriched ChIP-chip regions, detection of the absolute or relative transcriptional status of genes assessed by expression tiling experiments and, more importantly, it allows the integration of ChIP-chip and expression data. Results can be visualized instantly in a genomic context within the UCSC genome browser as graph-based custom tracks through Galaxy2. All generated and uploaded data can be stored within sessions and are easily shared with other users. AVAILABILITY: http://bio.ifom-ieo-campus.it/galaxy

Complex Loci in human and mouse genomes.

Mammalian genomes harbor a larger than expected number of complex loci, in which multiple genes are coupled by shared transcribed regions in antisense orientation and/or by bidirectional core promoters. To determine the incidence, functional significance, and evolutionary context of mammalian complex loci, we identified and characterized 5,248 cis-antisense pairs, 1,638 bidirectional promoters, and 1,153 chains of multiple cis-antisense and/or bidirectionally promoted pairs from 36,606 mouse transcriptional units (TUs), along with 6,141 cis-antisense pairs, 2,113 bidirectional promoters, and 1,480 chains from 42,887 human TUs. In both human and mouse, 25% of TUs resided in cis-antisense pairs, only 17% of which were conserved between the two organisms, indicating frequent species specificity of antisense gene arrangements. A sampling approach indicated that over 40% of all TUs might actually be in cis-antisense pairs, and that only a minority of these arrangements are likely to be conserved between human and mouse. Bidirectional promoters were characterized by variable transcriptional start sites and an identifiable midpoint at which overall sequence composition changed strand and the direction of transcriptional initiation switched. In microarray data covering a wide range of mouse tissues, genes in cis-antisense and bidirectionally promoted arrangement showed a higher probability of being coordinately expressed than random pairs of genes. In a case study on homeotic loci, we observed extensive transcription of nonconserved sequences on the noncoding strand, implying that the presence rather than the sequence of these transcripts is of functional importance. Complex loci are ubiquitous, host numerous nonconserved gene structures and lineage-specific exonification events, and may have a cis-regulatory impact on the member genes.

RaLP, a new member of the Src homology and collagen family, regulates cell migration and tumor growth of metastatic melanomas.

The Src homology and collagen (Src) family of adaptor proteins comprises six Shc-like proteins encoded by three loci in mammals (Shc, Rai, and Sli). Shc-like proteins are tyrosine kinase substrates, which regulate diverse signaling pathways and cellular functions, including Ras and proliferation (p52/p46Shc), phosphatidylinositol 3-kinase and survival (p54Rai), and mitochondrial permeability transition and apoptosis (p66Shc). Here, we report the identification, cloning, and sequence characterization of a new member of the Shc family that we termed RaLP. RaLP encodes a 69-kDa protein characterized by the CH2-PTB-CH1-SH2 modularity, typical of the Shc protein family, and expressed, among adult tissues, only in melanomas. Analysis of RaLP expression during the melanoma progression revealed low expression in normal melanocytes and benign nevi, whereas high levels of RaLP protein were found at the transition from radial growth phase to vertical growth phase and metastatic melanomas, when tumor cells acquire migratory competence and invasive potential. Notably, silencing of RaLP expression in metastatic melanomas by RNA interference reduced tumorigenesis in vivo. Analysis of RaLP in melanoma signal transduction pathways revealed that (a) when ectopically expressed in RaLP-negative melanocytes and nonmetastatic melanoma cells, it functions as a substrate of activated insulin-like growth factor-1 and epidermal growth factor receptors and increases Ras/mitogen-activated protein kinase (MAPK) signaling and cell migration, whereas (b) its silencing in RaLP-positive melanoma cells abrogates cell migration in vitro, without affecting MAPK signaling, suggesting that RaLP activates both Ras-dependent and Ras-independent migratory pathways in melanomas. These findings indicate that RaLP is a specific marker of metastatic melanomas, a critical determinant in the acquisition of the migratory phenotype by melanoma cells, and a potential target for novel anti-melanoma therapeutic strategies.

Development of Personalized Therapeutic Strategies by Targeting Actionable Vulnerabilities in Metastatic and Chemotherapy-Resistant Breast Cancer PDXs.

Human breast cancer is characterized by a high degree of inter-patients heterogeneity in terms of histology, genomic alterations, gene expression patterns, and metastatic behavior, which deeply influences individual prognosis and treatment response. The main cause of mortality in breast cancer is the therapy-resistant metastatic disease, which sets the priority for novel treatment strategies for these patients. In the present study, we demonstrate that Patient Derived Xenografts (PDXs) that were obtained from metastatic and therapy-resistant breast cancer samples recapitulate the wide spectrum of the disease in terms of histologic subtypes and mutational profiles, as evaluated by whole exome sequencing. We have integrated genomic and transcriptomic data to identify oncogenic and actionable pathways in each PDX. By taking advantage of primary short-term in vitro cultures from PDX tumors, we showed their resistance to standard chemotherapy (Paclitaxel), as seen in the patients. Moreover, we selected targeting drugs and analyzed PDX sensitivity to single agents or to combination of targeted and standard therapy on the basis of PDX-specific genomic or transcriptomic alterations. Our data demonstrate that PDXs represent a suitable model to test new targeting drugs or drug combinations and to prioritize personalized therapeutic regimens for pre-clinal and clinical tests.

Release of paused RNA polymerase II at specific loci favors DNA double-strand-break formation and promotes cancer translocations.

It is not clear how spontaneous DNA double-strand breaks (DSBs) form and are processed in normal cells, and whether they predispose to cancer-associated translocations. We show that DSBs in normal mammary cells form upon release of paused RNA polymerase II (Pol II) at promoters, 5' splice sites and active enhancers, and are processed by end-joining in the absence of a canonical DNA-damage response. Logistic and causal-association models showed that Pol II pausing at long genes is the main predictor and determinant of DSBs. Damaged introns with paused Pol II-pS5, TOP2B and XRCC4 are enriched in translocation breakpoints, and map at topologically associating domain boundary-flanking regions showing high interaction frequencies with distal loci. Thus, in unperturbed growth conditions, release of paused Pol II at specific loci and chromatin territories favors DSB formation, leading to chromosomal translocations.

p53 Loss in Breast Cancer Leads to Myc Activation, Increased Cell Plasticity, and Expression of a Mitotic Signature with Prognostic Value.

Loss of p53 function is invariably associated with cancer. Its role in tumor growth was recently linked to its effects on cancer stem cells (CSCs), although the underlying molecular mechanisms remain unknown. Here, we show that c-myc is a transcriptional target of p53 in mammary stem cells (MaSCs) and is activated in breast tumors as a consequence of p53 loss. Constitutive Myc expression in normal mammary cells leads to increased frequency of MaSC symmetric divisions, extended MaSC replicative-potential, and MaSC-reprogramming of progenitors, whereas Myc activation in breast cancer is necessary and sufficient to maintain the expanding pool of CSCs. Concomitant p53 loss and Myc activation trigger the expression of 189 mitotic genes, which identify patients at high risk of mortality and relapse, independently of other risk factors. Altogether, deregulation of the p53:Myc axis in mammary tumors increases CSC content and plasticity and is a critical determinant of tumor growth and clinical aggressiveness.

Delocalization and destabilization of the Arf tumor suppressor by the leukemia-associated NPM mutant.

One third of acute myeloid leukemias (AMLs) are characterized by the aberrant cytoplasmic localization of nucleophosmin (NPM) due to mutations within its putative nucleolar localization signal. NPM mutations are mutually exclusive with major AML-associated chromosome rearrangements and are frequently associated with a normal karyotype, suggesting that they are critical during leukemogenesis. The underlying molecular mechanisms are, however, unknown. NPM is a nucleocytoplasmic shuttling protein that has been implicated in several cellular processes, including ribosome biogenesis, centrosome duplication, cell cycle progression, and stress response. It has been recently shown that NPM is required for the stabilization and proper nucleolar localization of the tumor suppressor p19(Arf). We report here that the AML-associated NPM mutant localizes mainly in the cytoplasm due to an alteration of its nucleus-cytoplasmic shuttling equilibrium, forms a direct complex with p19(Arf), but is unable to protect it from degradation. Consequently, cells or leukemic blasts expressing the NPM mutant have low levels of cytoplasmic Arf. Furthermore, we show that expression of the NPM mutant reduces the ability of Arf to initiate a p53 response and to induce cell cycle arrest. Inactivation of p19(Arf), a key regulator of the p53-dependent cellular response to oncogene expression, might therefore contribute to leukemogenesis in AMLs with mutated NPM.

Epigenomic profiling of archived FFPE tissues by enhanced PAT-ChIP (EPAT-ChIP) technology.

BACKGROUND: The introduction of pathology tissue-chromatin immunoprecipitation (PAT-ChIP), a technique allowing chromatin immunoprecipitation (ChIP) from formalin-fixed paraffin-embedded (FFPE) tissues, has extended the application of chromatin studies to clinical patient samples. However, extensive crosslinking introduced during routine tissue fixation of clinical specimens may hamper the application of PAT-ChIP to genome-wide studies (PAT-ChIP-Seq) from archived tissue samples. The reduced efficiency in chromatin extraction from over-fixed formalin archival samples is the main hurdle to overcome, especially when low abundant epigenetic marks (e.g., H3K4me3) are investigated. RESULTS: We evaluated different modifications of the original PAT-ChIP protocol to improve chromatin isolation from FFPE tissues. With this aim, we first made extensive usage of a normal human colon specimen fixed at controlled conditions (24 h, 48 h, and 72 h) to mimic the variability of tissue fixation that is most frequently found in archived samples. Different conditions of chromatin extraction were tested applying either diverse sonication protocols or heat-mediated limited reversal of crosslinking (LRC). We found that, if compared with canonical PAT-ChIP protocol, LRC strongly increases chromatin extraction efficiency, especially when 72-h fixed FFPE samples are used. The new procedure, that we named enhanced PAT-ChIP (EPAT-ChIP), was then applied at genome-wide level using an archival sample of invasive breast carcinoma to investigate H3K4me3, a lowly abundant histone modification, and H3K27me3 and H3K27ac, two additional well-known histone marks. CONCLUSIONS: EPAT-ChIP procedure improves the efficiency of chromatin isolation from FFPE samples allowing the study of long time-fixed specimens (72 h), as well as the investigation of low distributed epigenetic marks (e.g., H3K4me3) and the analysis of multiple histone marks from low amounts of starting material. We believe that EPAT-ChIP will facilitate the application of chromatin studies to archived pathology samples, thus contributing to extend the current understanding of cancer epigenomes and enabling the identification of clinically useful tumor biomarkers.

PREP1 tumor suppressor protects the late-replicating DNA by controlling its replication timing and symmetry.

The synthesis of middle-to-late-replicating DNA can be affected independently of the rest of the genome by down-regulating the tumor suppressor PREP1 (PKNOX1). Indeed, DNA combing shows that PREP1 down-regulation affects DNA replication rate, increases the number of simultaneously firing origins and the asymmetry of DNA replication, leading to DNA damage. Genome-wide analysis of replication timing by Repli-seq shows that, upon PREP1 down-regulation, 25% of the genome is replicated earlier in the S-phase. The targeted DNA sequences correspond to Lamin-Associated Domains (LADs), and include late-replicating (LRRs) and temporal transition regions (TTRs). Notably, the distribution of PREP1 DNA binding sites and of its target genes indicates that DNA replication defects are independent of the overall PREP1 transcriptional activity. Finally, PREP1 down-regulation causes a substantial decrease in Lamin B1 levels. This suggests that DNA is released from the nuclear lamina earlier than in the control cells and is available for replication, thus explaining timing defects and DNA damage.This is the first evidence that the replication timing of a specific fraction of the human genome is affected by PREP1 tumor suppressor. This previously unknown function might significantly contribute to the genomic instability observed in human tumors.