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The precise spatiotemporal expression of the hematopoietic ETS transcription factor PU.1, a key determinant of hematopoietic cell fates, is tightly regulated at the chromatin level. However, how chromatin signatures are linked to this dynamic expression pattern across different blood cell lineages remains uncharacterized. Here, we performed an in-depth analysis of the relationships between gene expression, chromatin structure, 3D architecture, and trans-acting factors at PU.1 cis-regulatory elements (PCREs). By identifying phylogenetically conserved DNA elements within chromatin-accessible regions in primary human blood lineages, we discovered multiple novel candidate PCREs within the upstream region of the human PU.1 locus. A subset of these elements localizes within an 8-kb-wide cluster exhibiting enhancer features, including open chromatin, demethylated DNA, enriched enhancer histone marks, present enhancer RNAs, and PU.1 occupation, presumably mediating PU.1 autoregulation. Importantly, we revealed the presence of a common 35-kb-wide CTCF-flanked insulated neighborhood that contains the PCRE cluster (PCREC), forming a chromatin territory for lineage-specific and PCRE-mediated chromatin interactions. These include functional PCRE-promoter interactions in myeloid and B cells that are absent in erythroid and T cells. By correlating chromatin structure and 3D architecture with PU.1 expression in various lineages, we were able to attribute enhancer versus silencer functions to individual elements. Our findings provide mechanistic insights into the interplay between dynamic chromatin structure and 3D architecture in the chromatin regulation of PU.1 expression. This study lays crucial groundwork for additional experimental studies that validate and dissect the role of PCREs in epigenetic regulation of normal and malignant hematopoiesis. Main points An unbiased, multiomics comparison between multiple blood cell types identified novel PU.1 cis regulatory elements with specific chromatin signatures. A 35-kb insulated neighborhood forms a territory for lineage-specific interactions involving an 8-kb PU.1 cis regulatory element cluster in 3D chromatin space.

The Ets transcription factor PU.1 is essential for inducing the differentiation of monocytes, macrophages, and B cells in fetal liver and adult bone marrow. PU.1 controls hematopoietic differentiation through physical interactions with other transcription factors, such as C/EBPα and the AP-1 family member c-Jun. We found that PU.1 recruits c-Jun to promoters without the AP-1 binding sites. To address the functional importance of this interaction, we generated PU.1 point mutants that do not bind c-Jun while maintaining normal DNA binding affinity. These mutants lost the ability to transactivate a target reporter that requires a physical PU.1-c-Jun interaction, and did not induce monocyte/macrophage differentiation of PU.1-deficient cells. Knock-in mice carrying these point mutations displayed an almost complete block in hematopoiesis and perinatal lethality. While the PU.1 mutants were expressed in hematopoietic stem and early progenitor cells, myeloid differentiation was severely blocked, leading to an almost complete loss of mature hematopoietic cells. Differentiation into mature macrophages could be restored by expressing PU.1 mutant fused to c-Jun, demonstrating that a physical PU.1-c-Jun interaction is crucial for the transactivation of PU.1 target genes required for myeloid commitment and normal PU.1 function in vivo during macrophage differentiation. The transcription factor PU.1 recruits c-Jun as a co-activator to promoters without AP-1 binding sites, and mice with point mutations in PU.1 that disrupts the interaction between PU1 and c-Jun have defects in PU.1 dependent blood lineages, including macrophages and B cells.

Nutritional intake impacts the human epigenome by directing epigenetic pathways in normal cell development via as yet unknown molecular mechanisms. Consequently, imbalance in the nutritional intake is able to dysregulate the epigenetic profile and drive cells towards malignant transformation. Here we present a novel epigenetic effect of the essential nutrient, NAD. We demonstrate that impairment of DNMT1 enzymatic activity by NAD-promoted ADP-ribosylation leads to demethylation and transcriptional activation of the CEBPA gene, suggesting the existence of an unknown NAD-controlled region within the locus. In addition to the molecular events, NAD- treated cells exhibit significant morphological and phenotypical changes that correspond to myeloid differentiation. Collectively, these results delineate a novel role for NAD in cell differentiation, and indicate novel nutri-epigenetic strategies to regulate and control gene expression in human cells.

Advancements in genome sequencing technologies have uncovered the multifaceted roles of long non-coding RNAs (lncRNAs) in human cells. Recent discoveries have identified lncRNAs as major players in gene regulatory pathways, highlighting their pivotal role in human cell growth and development. Their dysregulation is implicated in the onset of genetic disorders and age-related diseases, including cancer. Specifically, they have been found to orchestrate molecular mechanisms impacting epigenetics, including DNA methylation and hydroxymethylation, histone modifications, and chromatin remodeling, thereby significantly influencing gene expression. This review provides an overview of the current knowledge on lncRNA-mediated epigenetic regulation of gene expression, emphasizing the biomedical implications of lncRNAs in the development of different types of cancers and genetic diseases.

Coordinated initiation of DNA replication is essential to ensure efficient and timely DNA synthesis. Yet the molecular determinants that confer origin selectivity in mammalian cells remain incompletely defined. Herein, we present data demonstrating a pivotal role for RNAs transcribed in the proximity of actively replicating gene loci. We show that RNAs aNChoring ORC1 (ANCORs) to the histone variant H2A.Z facilitate origin firing during DNA replication. This ANCOR-H2A.Z interaction appears to be essential for cells to duplicate their genetic material. Widespread and locus-specific perturbations of these transcripts correlate with anomalous replication patterns and a notable loss of the H2A.Z replicative marker at the origin site. Collectively, we present a previously undescribed RNA-mediated mechanism that is associated with the generation of active replication origins in mammalian cells. Our findings delineate a strategy to modulate the origins of replication in human cells at a local and global level, with potentially broad biomedical implications.

High expression of the myeloid master ETS transcription factor PU.1 drives the development of monocyte/macrophage (Mono/MΦ), a crucial cellular component of the innate immune system. Disruptions in normal expression patterns of PU.1 are linked to a variety myeloid malignancy and immune diseases. It is evidenced that PU.1 binds to and modulates enhancers of several myeloid genes. While noncoding RNAs transcribed from noncoding genes at the enhancers are increasingly reported to be involved in enhancer regulation, the crosstalk between PU.1 and noncoding RNAs in enhancer-mediated myeloid gene regulation in Mono/MΦ differentiation and immune response has not been systematically investigated. In this study, we interrogated the PU.1-mediated transcriptome and cistrome with our comprehensive collection of putative and verified enhancers. Among a repertoire of noncoding genes present at PU.1-bound enhancers, we discovered that PU.1 acts as a potent transcription factor inducer of the noncoding RNA , which we previously identified as an RNA inducer of PU.1. The genomic region within the locus occupied by PU.1 is characterized by the epigenetic features of a myeloid-specific super-enhancer. Targeted disruption of the PU.1-binding motifs resulted in the downregulation of promoter activity. Depletion of reduced the expression of Mono/MΦ cell markers as well as the transcriptional program associated with Mono/MΦ differentiation Mono/MΦ innate defense mechanisms, including phagocytosis, antimicrobial activity, and chemoattractant cytokine production. induces Mono/MΦ phagocytic activities. Collectively, our findings indicate that PU.1 and enhancer RNA are biomolecular components of an unidentified feed-forward loop that promotes their mutual expression, contributing to Mono/MΦ differentiation and innate immune functions. The identification of the PU.1/ regulatory circuit provides valuable insights into the mechanisms underlying cell-type and gene-specific enhancer activity and Mono/MΦ biology, as well as significant implications for advancing our understanding of immune diseases and myeloid malignancies.

The precise spatio-temporal expression of the hematopoietic ETS transcription factor that determines the hematopoietic cell fates is tightly regulated at the chromatin level. However, it remains elusive as to how chromatin signatures are linked to this dynamic expression pattern across blood cell lineages. Here we performed an unbiased and in-depth analysis of the relationship between human expression, the presence of trans-acting factors, and 3D architecture at various cis-regulatory elements (CRE) proximal to the locus. We identified multiple novel CREs at the upstream region of the gene following an integrative inspection for conserved DNA elements at the chromatin-accessible regions in primary human blood lineages. We showed that a subset of CREs localize within a 10 kb-wide cluster that exhibits that exhibit molecular features of a myeloid-specific super-enhancer involved in mediating autoregulation, including open chromatin, unmethylated DNA, histone enhancer marks, transcription of enhancer RNAs, and occupancy of the PU.1 protein itself. Importantly, we revealed the presence of common 35-kb-wide CTCF-bound insulated neighborhood that contains the CRE cluster, forming the chromatin territory for lineage-specific and CRE-mediated chromatin interactions. These include functional CRE-promoter interactions in myeloid and B cells but not in erythroid and T cells. Our findings also provide mechanistic insights into the interplay between dynamic chromatin structure and 3D architecture in defining certain CREs as enhancers or silencers in chromatin regulation of expression. The study lays the groundwork for further examination of CREs as well as epigenetic regulation in malignant hematopoiesis.

Nutritional intake impacts the human epigenome by directing epigenetic pathways in normal cell development via as yet unknown molecular mechanisms. Consequently, imbalance in the nutritional intake is able to dysregulate the epigenetic profile and drive cells towards malignant transformation. Herein, we present a novel epigenetic effect of the essential nutrient, NAD. We demonstrate that impairment of DNMT1 enzymatic activity by NAD-promoted ADP-ribosylation, leads to demethylation and transcriptional activation of CEBPA gene, suggesting the existence of an unknown NAD-controlled region within the locus. In addition to the molecular events, NAD treated cells exhibit significant morphological and phenotypical changes that correspond to myeloid differentiation. Collectively, these results delineate a novel role for NAD in cell differentiation and indicate novel nutri-epigenetic strategy to regulate and control gene expression in human cells. Competing Interest Statement SSK reports research grants and honorarium from Boehringer Ingelheim, grants from Taiho Pharmaceutical and MiNA therapeutics, and honorarium from Pfizer, Ono, Chugai, Astra Zeneca, and Roche outside the submitted work. The other authors declare no conflict of interests.

The mechanism underlying cell type-specific gene induction conferred by ubiquitous transcription factors as well as disruptions caused by their chimeric derivatives in leukemia is not well understood. Here we investigate whether RNAs coordinate with transcription factors to drive myeloid gene transcription. In an integrated genome-wide approach surveying for gene loci exhibiting concurrent RNA- and DNA-interactions with the broadly expressed transcription factor RUNX1, we identified the long noncoding RNA LOUP. This myeloid-specific and polyadenylated lncRNA induces myeloid differentiation and inhibits cell growth, acting as a transcriptional inducer of the myeloid master regulator PU.1. Mechanistically, LOUP recruits RUNX1 to both the PU.1 enhancer and the promoter, leading to the formation of an active chromatin loop. In t(8;21) acute myeloid leukemia, wherein RUNX1 is fused to ETO, the resulting oncogenic fusion protein RUNX1-ETO limits chromatin accessibility at the LOUP locus, causing inhibition of LOUP and PU.1 expression. These findings highlight the important role of the interplay between cell type-specific RNAs and transcription factors as well as their oncogenic derivatives in modulating lineage-gene activation and raise the possibility that RNA regulators of transcription factors represent alternative targets for therapeutic development. Competing Interest Statement The authors have declared no competing interest.

Coordinated initiation of DNA replication is essential to ensure efficient and timely DNA synthesis. Yet, the mechanism governing the initiation process in eukaryotic cells remains elusive. Here, we present data demonstrating a novel feature of RNAs transcribed in the proximity of actively replicating gene loci. We show that S-phAse-RNAs aNChoring ORC1 (ANCORs) to the histone variant H2A.Z are licensors of the DNA replication process. The concomitant ANCOR-H2A.Z interaction is essential for the cells to initiate duplication of their genetic heritage. Widespread and locus-specific perturbations of these transcripts correlate with anomalous replication patterns and loss of the replicative marker at the origin site. Collectively, we unveil an RNA-mediated mechanism as the missing link for the generation of active replication origins and delineate a potential strategy to modulate replication in human cells at a local and global level.Competing Interest StatementThe authors have declared no competing interest.Footnotes* Manuscript Updated Figures Updated Authors Updated Supplemental Figures updated