Distal Elements Research Articles

Chromatin interaction maps identify oncogenic targets of enhancer duplications in cancer.

As a major type of structural variants, tandem duplication plays a critical role in tumorigenesis by increasing oncogene dosage. Recent work has revealed that noncoding enhancers are also affected by duplications leading to the activation of oncogenes that are inside or outside of the duplicated regions. However, the prevalence of enhancer duplication and the identity of their target genes remains largely unknown in the cancer genome. Here, by analyzing whole-genome sequencing data in a non-gene-centric manner, we identify 881 duplication hotspots in 13 major cancer types, most of which do not contain protein-coding genes. We show that the hotspots are enriched with distal enhancer elements and are highly lineage-specific. We develop a HiChIP-based methodology that navigates enhancer-promoter contact maps to prioritize the target genes for the duplication hotspots harboring enhancer elements. The methodology identifies many novel enhancer duplication events activating oncogenes such as ESR1, FOXA1, GATA3, GATA6, TP63, and VEGFA, as well as potentially novel oncogenes such as GRHL2, IRF2BP2, and CREB3L1 In particular, we identify a duplication hotspot on Chromosome 10p15 harboring a cluster of enhancers, which skips over two genes, through a long-range chromatin interaction, to activate an oncogenic isoform of the NET1 gene to promote migration of gastric cancer cells. Focusing on tandem duplications, our study substantially extends the catalog of noncoding driver alterations in multiple cancer types, revealing attractive targets for functional characterization and therapeutic intervention.

Inhibition of Renin Expression Is Regulated by an Epigenetic Switch From an Active to a Poised State.

Renin-expressing cells are myoendocrine cells crucial for the maintenance of homeostasis. Renin is regulated by cAMP, p300 (histone acetyltransferase p300)/CBP (CREB-binding protein), and Brd4 (bromodomain-containing protein 4) proteins and associated pathways. However, the specific regulatory changes that occur following inhibition of these pathways are not clear. We treated As4.1 cells (tumoral cells derived from mouse juxtaglomerular cells that constitutively express renin) with 3 inhibitors that target different factors required for renin transcription: H-89-dihydrochloride, PKA (protein kinase A) inhibitor; JQ1, Brd4 bromodomain inhibitor; and A-485, p300/CBP inhibitor. We performed assay for transposase-accessible chromatin with sequencing (ATAC-seq), single-cell RNA sequencing, cleavage under targets and tagmentation (CUT&Tag), and chromatin immunoprecipitation sequencing for H3K27ac (acetylation of lysine 27 of the histone H3 protein) and p300 binding on biological replicates of treated and control As4.1 cells. In response to each inhibitor, Ren1 expression was significantly reduced and reversible upon washout. Chromatin accessibility at the Ren1 locus did not markedly change but was globally reduced at distal elements. Inhibition of PKA led to significant reductions in H3K27ac and p300 binding specifically within the Ren1 super-enhancer region. Further, we identified enriched TF (transcription factor) motifs shared across each inhibitory treatment. Finally, we identified a set of 9 genes with putative roles across each of the 3 renin regulatory pathways and observed that each displayed differentially accessible chromatin, gene expression, H3K27ac, and p300 binding at their respective loci. Inhibition of renin expression in cells that constitutively synthesize and release renin is regulated by an epigenetic switch from an active to poised state associated with decreased cell-cell communication and an epithelial-mesenchymal transition. This work highlights and helps define the factors necessary for renin cells to alternate between myoendocrine and contractile phenotypes.

TIANA: transcription factors cooperativity inference analysis with neural attention

BackgroundGrowing evidence suggests that distal regulatory elements are essential for cellular function and states. The sequences within these distal elements, especially motifs for transcription factor binding, provide critical information about the underlying regulatory programs. However, cooperativities between transcription factors that recognize these motifs are nonlinear and multiplexed, rendering traditional modeling methods insufficient to capture the underlying mechanisms. Recent development of attention mechanism, which exhibit superior performance in capturing dependencies across input sequences, makes them well-suited to uncover and decipher intricate dependencies between regulatory elements.ResultWe present Transcription factors cooperativity Inference Analysis with Neural Attention (TIANA), a deep learning framework that focuses on interpretability. In this study, we demonstrated that TIANA could discover biologically relevant insights into co-occurring pairs of transcription factor motifs. Compared with existing tools, TIANA showed superior interpretability and robust performance in identifying putative transcription factor cooperativities from co-occurring motifs.ConclusionOur results suggest that TIANA can be an effective tool to decipher transcription factor cooperativities from distal sequence data. TIANA can be accessed through: https://github.com/rzzli/TIANA.

Myddosomes in Toll-like receptor signaling-one to bind and rule them all.

Toll-like receptors (TLRs) are innate immune sensors for the presence of pathogens and endogenous danger signals. TLR activation results in conserved intracellular signaling events that orchestrate inflammation and antimicrobial defense. While the identity and interplay of key TLR signaling components are well established, how these largely cytosolic proteins are physically connected is not well understood. For the activation of conserved intracellular signaling events, most TLRs engage the adapter MyD88 (myeloid differentiation primary response 88), which assembles into higher-order protein complexes, myddosomes. In their recent publication, Fisch etal. present evidence that oligomeric myddosomes detach from initiating TLRs and evolve into larger scaffolds that dynamically assemble not only proximal but also distal cytosolic elements required to execute the entire cascade of the TLR-MyD88 signaling pathway. Coinciding with decline in TLR signaling over time, myddosomes progressively recruit autophagy machinery that mediates myddosome clearance. These findings expand the current understanding of TLR signaling by positioning myddosomes as the central structural element that physically assembles the key executors and regulators of TLR-MyD88-dependent intracellular signaling cascades.

CREB activates the MafA promoter through proximal E-boxes and a CCAAT motif in pancreatic β-cells.

MafA is a key transcriptional regulator of pancreatic islet β-cell function. Its target genes include those encoding preproinsulin and the glucose transporter Glut2 (Slc2a2); thus, MafA function is essential for glucose-stimulated insulin secretion. Expression levels of MafA are reduced in β-cells of diabetic mouse models and human subjects, suggesting that β-cell dysfunction associated with type 2 diabetes is attributable to the loss of MafA. On the other hand, MafA is transcriptionally upregulated by incretin hormones through activation of CREB and its co-activator CRTC2. β-cell-specific expression of MafA relies on a distal enhancer element. However, the precise mechanism by which CREB-CRTC2 regulates the enhancer and proximal promoter regions of MafA remains unclear. In this report, we analyzed previously published ChIP-seq data and found that CREB and NeuroD1, a β-cell-enriched transactivator, bound to both the promoter and enhancer regions of human MAFA. A series of reporter assays revealed that CREB activated the enhancer through a conserved cAMP-responsive element (CRE) but stimulated MAFA promoter activity even when the putative CRE was deleted. Two E-box elements and a CCAAT motif, which bind NeuroD1 and ubiquitous NF-Y transcription factors, respectively, were necessary for transcriptional activation of the MAFA promoter by CREB. Genome-wide analysis of CREB-bound loci in β-cells revealed that they were enriched with CCAAT motifs. Furthermore, promoter analysis of the Isl1 gene encoding a β-cell-enriched transcription factor revealed that a CRE-like element and two CCAAT motifs, but not the E-box, were necessary for activation by CREB. These results provide clues to elucidate the detailed mechanism by which CREB regulates MafA as well as β-cell-specific genes.

EPInformer: a scalable deep learning framework for gene expression prediction by integrating promoter-enhancer sequences with multimodal epigenomic data.

Transcriptional regulation, critical for cellular differentiation and adaptation to environmental changes, involves coordinated interactions among DNA sequences, regulatory proteins, and chromatin architecture. Despite extensive data from consortia like ENCODE, understanding the dynamics of cis-regulatory elements (CREs) in gene expression remains challenging. Deep learning is a powerful tool for learning gene expression and epigenomic signals from DNA sequences, exhibiting superior performance compared to conventional machine learning approaches. However, even the most advanced deep learning-based methods may fall short in capturing the regulatory effects of distal elements such as enhancers, limiting their predictive accuracy. In addition, these methods may require significant resources to train or to adapt to newly generated data. To address these challenges, we present EPInformer, a scalable deep-learning framework for predicting gene expression by integrating promoter-enhancer interactions with their sequences, epigenomic signals, and chromatin contacts. Our model outperforms existing gene expression prediction models in rigorous cross-chromosome validation, accurately recapitulates enhancer-gene interactions validated by CRISPR perturbation experiments, and identifies crucial transcription factor motifs within regulatory sequences. EPInformer is available as open-source software at https://github.com/pinellolab/EPInformer.

Direct Inhibition of the YAP : TEAD Interaction: An Unprecedented Drug Discovery Challenge.

The Hippo pathway, which is key in organ morphogenesis, is frequently deregulated in cancer. The TEAD (TEA domain family member) transcription factors are the most distal elements of this pathway, and their activity is regulated by proteins such as YAP (Yes-associated protein). The identification of inhibitors of the YAP : TEAD interaction is one approach to develop novel anticancer drugs: the first clinical candidate (IAG933) preventing the association between these two proteins by direct competition has just been reported. The discovery of this molecule was particularly challenging because the interface between these two proteins is large (~3500 Å2 buried in complex formation) and made up of distinct contact areas. The most critical of these involves an omega-loop (Ω-loop), a secondary structure element rarely found in protein-protein interactions. This review summarizes how the knowledge gained from structure-function studies of the interaction between the Ω-loop of YAP and TEAD was used to devise the strategy to identify potent low-molecular weight compounds that show a pronounced anti-tumor effect.

Evolutionary change of crystallographic orientation and coccolith morphology: Neogene-Quaternary Umbilicosphaera (Prymnesiophyceae) lineage

The crystallographic orientation and its relationship to the morphology of coccoliths were investigated for the Neogene-Quaternary calcareous nannoplankton Umbilicosphaera lineage using scanning electron microscopy (SEM) and electron back-scattered diffraction (EBSD). The c-axis of the calcite forming the distal shield elements was inclined upward at 66–68° (U. sibogae), 65–68° (U. foliosa), 57–58° (U. rotula), and 55–57° (U. patera) from the coccolith plane. Accordingly, the outward dip angle of one of the {101¯4} faces forming the surface of the distal shield of U. patera coccolith was shallower than those of U. sibogae and U. foliosa, explaining the nearly flat distal shield and the steep inner slope, formed by another equivalent {101¯4} face, around the central opening of U. patera. Our results showed that the evolution from U. rotula to U. patera during the Late Miocene was not accompanied by a change in crystallographic orientation. In contrast, the evolution from U. patera to U. sibogae and U. foliosa during the Pliocene was accompanied by a rotation of the orientation. The crystallographic orientation of calcite nuclei on the baseplate with a combination of other factors would have resulted in species-specific differences in shield shape and suture lines within the same phylogeny, consequently producing morphological diversity in the coccolith throughout geological time.

Soft-Tissue Augmentation Around Dental Implants by a New Laser-Aided Pouch Roll Technique: Volumetric and Clinical Evaluation After 1 Year.

The ultimate goal in implantology is to restore the whole tooth-gingival complex in a fashion that cannot be distinguished from the rest of the natural dentition. This study assesses the volumetric and clinical changes in vestibular gingival soft tissues, crucial for satisfactory engraftment and esthetic results, upon treatment with laser-aided pouch roll augmentation in second-stage surgery for dental implant uncovering. Twelve patients with mild ridge deficiencies in 16 edentulous sites, including distal elements, were enrolled and reevaluated for up to 1 year. Digital impressions, taken with an intraoral laser scanner and software, were analyzed before (day 0) and after (month 12) treatment. The digital STL files were superimposed to assess volumetric and linear dimensional variations in selected peri-implant regions of interest by 3D analysis software. Clinical periodontal parameters (probing depth [PD], bleeding on probing [BoP], plaque index [PI]) and subjective patient-reported outcomes were also evaluated. In all patients, the applied technique induced a substantial increase in the volume of the vestibular peri-implant gingiva at 12-month follow-up (range, 24%-69%, mean 40.4%) with respect to day 0. The gingival mucosa appeared normal at both inspection and evaluation of the periodontal parameters (PD 2.7 ± 1 mm; BoP 0.11 ± 0.2 seconds; PI 0.19 ± 0). Patients' satisfaction with perceived pain/discomfort and esthetic outcome was high. These findings indicate that laser-aided pouch roll flap is a safe, patient-liked procedure whose long-term effectiveness has been objectively demonstrated by volumetric and clinical assessment.

Activity-driven chromatin organization during interphase: Compaction, segregation, and entanglement suppression

In mammalian cells, the cohesin protein complex is believed to translocate along chromatin during interphase to form dynamic loops through a process called active loop extrusion. Chromosome conformation capture and imaging experiments have suggested that chromatin adopts a compact structure with limited interpenetration between chromosomes and between chromosomal sections. We developed a theory demonstrating that active loop extrusion causes the apparent fractal dimension of chromatin to cross-over between two and four at contour lengths on the order of 30 kilo-base pairs. The anomalously high fractal dimension [Formula: see text] is due to the inability of extruded loops to fully relax during active extrusion. Compaction on longer contour length scales extends within topologically associated domains (TADs), facilitating gene regulation by distal elements. Extrusion-induced compaction segregates TADs such that overlaps between TADs are reduced to less than 35% and increases the entanglement strand of chromatin by up to a factor of 50 to several Mega-base pairs. Furthermore, active loop extrusion couples cohesin motion to chromatin conformations formed by previously extruding cohesins and causes the mean square displacement of chromatin loci during lag times ([Formula: see text]) longer than tens of minutes to be proportional to [Formula: see text]. We validate our results with hybrid molecular dynamics-Monte Carlo simulations and show that our theory is consistent with experimental data. This work provides a theoretical basis for the compact organization of interphase chromatin, explaining the physical reason for TAD segregation and suppression of chromatin entanglements which contribute to efficient gene regulation.

CRISPR-dCas13d-based deep screening of proximal and distal splicing-regulatory elements

Pre-mRNA splicing, a key process in gene expression, can be therapeutically modulated using various drug modalities, including antisense oligonucleotides (ASOs). However, determining promising targets is hampered by the challenge of systematically mapping splicing-regulatory elements (SREs) in their native sequence context. Here, we use the catalytically inactive CRISPR-RfxCas13d RNA-targeting system (dCas13d/gRNA) as a programmable platform to bind SREs and modulate splicing by competing against endogenous splicing factors. SpliceRUSH, a high-throughput screening method, was developed to map SREs in any gene of interest using a lentivirus gRNA library that tiles the genetic region, including distal intronic sequences. When applied to SMN2, a therapeutic target for spinal muscular atrophy, SpliceRUSH robustly identifies not only known SREs but also a previously unknown distal intronic SRE, which can be targeted to alter exon 7 splicing using either dCas13d/gRNA or ASOs. This technology enables a deeper understanding of splicing regulation with applications for RNA-based drug discovery.

KSHV genome harbors both constitutive and lytically induced enhancers.

Kaposi's sarcoma-associated herpesvirus (KSHV) belongs to the gamma-herpesvirus family and is a well-known human oncogenic virus. In infected cells, the viral genome of 165 kbp is circular DNA wrapped in chromatin. The tight control of gene expression is critical for latency, the transition into the lytic phase, and the development of viral-associated malignancies. Distal cis-regulatory elements, such as enhancers and silencers, can regulate gene expression in a position- and orientation-independent manner. Open chromatin is another characteristic feature of enhancers. To systematically search for enhancers, we cloned all the open chromatin regions in the KSHV genome downstream of the luciferase gene and tested their enhancer activity in infected and uninfected cells. A silencer was detected upstream of the latency-associated nuclear antigen promoter. Two constitutive enhancers were identified in the K12p-OriLyt-R and ORF29 Intron regions, where ORF29 Intron is a tissue-specific enhancer. The following promoters: OriLyt-L, PANp, ALTp, and the terminal repeats (TRs) acted as lytically induced enhancers. The expression of the replication and transcription activator (RTA), the master regulator of the lytic cycle, was sufficient to induce the activity of lytic enhancers in uninfected cells. We propose that the TRs that span about 24 kbp region serve as a "viral super-enhancer" that integrates the repressive effect of the latency-associated nuclear antigen (LANA) with the activating effect of RTA. Utilizing CRISPR activation and interference techniques, we determined the connections between these enhancers and their regulated genes. The silencer and enhancers described here provide an additional layer to the complex gene regulation of herpesviruses.IMPORTANCEIn this study, we performed a systematic functional assay to identify cis-regulatory elements within the genome of the oncogenic herpesvirus, Kaposi's sarcoma-associated herpesvirus (KSHV). Similar to other herpesviruses, KSHV presents both latent and lytic phases. Therefore, our assays were performed in uninfected cells, during latent infection, and under lytic conditions. We identified two constitutive enhancers, one of which seems to be a tissue-specific enhancer. In addition, four lytically induced enhancers, which are all responsive to the replication and transcription activator (RTA), were identified. Furthermore, a silencer was identified between the major latency promoter and the lytic gene locus. Utilizing CRISPR activation and interference techniques, we determined the connections between these enhancers and their regulated genes. The terminal repeats, spanning a region of about 24 kbp, seem like a "viral super-enhancer" that integrates the repressive effect of the latency-associated nuclear antigen (LANA) with the activating effect of RTA to regulate latency to lytic transition.

Integrative analysis of transcriptomic and epigenomic data reveals distinct patterns for developmental and housekeeping gene regulation

BackgroundRegulation of transcription is central to the emergence of new cell types during development, and it often involves activation of genes via proximal and distal regulatory regions. The activity of regulatory elements is determined by transcription factors (TFs) and epigenetic marks, but despite extensive mapping of such patterns, the extraction of regulatory principles remains challenging.ResultsHere we study differentially and similarly expressed genes along with their associated epigenomic profiles, chromatin accessibility and DNA methylation, during lineage specification at gastrulation in mice. Comparison of the three lineages allows us to identify genomic and epigenomic features that distinguish the two classes of genes.We show that differentially expressed genes are primarily regulated by distal elements, while similarly expressed genes are controlled by proximal housekeeping regulatory programs. Differentially expressed genes are relatively isolated within topologically associated domains, while similarly expressed genes tend to be located in gene clusters. Transcription of differentially expressed genes is associated with differentially open chromatin at distal elements including enhancers, while that of similarly expressed genes is associated with ubiquitously accessible chromatin at promoters.ConclusionBased on these associations of (linearly) distal genes’ transcription start sites (TSSs) and putative enhancers for developmental genes, our findings allow us to link putative enhancers to their target promoters and to infer lineage-specific repertoires of putative driver transcription factors, within which we define subgroups of pioneers and co-operators.

New insights into the functional morphology of calceoli in Amphipoda (Crustacea)

Calceoli are putative sensory organs which are known in limnic, marine, and subterranean amphipods for almost 200 years. Despite an otherwise comprehensive understanding of the sensory organs of crustaceans, we still have not unambiguously understood their function. Since calceoli are mainly found on the antennae of male animals, previous studies suggested a chemosensory function mainly related to reproduction. Here, we use a combination of light and electron microscopic techniques to examine the calceoli of Gammarus locusta (Linnaeus 1758) and Oediceroides calmani (Walker 1906), to provide an overview over these structures, and in addition reveal nervous tissue in close proximity to the calceoli. The calceoli of both species are cuticular structures and consist of proximal and distal elements, a stalk and a receptacle that connects both regions. The two studied calceoli differ in the structure of their proximal and distal element, as well as in their receptacle. This study provides new insight into the functional morphology of the antenna and calceolus. Histological sections through the antennae and the calceoli indicate that the calceoli might possess a mechanosensory function.

Alteration of chromosome structure impacts gene expressions implicated in pancreatic ductal adenocarcinoma cells

BackgroundPancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with a five-year survival rate of approximately 10%. Genetic mutations are pivotal drivers in PDAC pathogenesis, but recent investigations also revealed the involvement of non-genetic alterations in the disease development. In this study, we undertook a multi-omics approach, encompassing ATAC-seq, RNA-seq, ChIP-seq, and Hi-C methodologies, to dissect gene expression alterations arising from changes in chromosome accessibility and chromatin three-dimensional interactions in PDAC.ResultsOur findings indicate that chromosomal structural alterations can lead to abnormal expressions on key genes during PDAC development. Notably, overexpression of oncogenes FGFR2, FOXA2, CYP2R1, and CPOX can be attributed to the augmentation of promoter accessibility, coupled with long-range interactions with distal elements. Additionally, our findings indicate that chromosomal structural alterations caused by genomic instability can lead to abnormal expressions in PDACs. As an example, by analyzing chromosomal changes, we identified a putative oncogenic gene, LPAR1, which shows upregulated expression in both PDAC cell lines and clinical samples. The overexpression is correlated with alterations in LPAR1-associated 3D genome structure and chromatin state. We further demonstrated that high LPAR1 activity is required for enhanced PDAC cell migration in vitro.ConclusionsCollectively, our findings reveal that the chromosomal conformational alterations, in addition to the well-known genetic mutations, are critical for PDAC tumorigenesis.

Pathogenic variants in CRX have distinct cis-regulatory effects on enhancers and silencers in photoreceptors.

Dozens of variants in the gene for the homeodomain transcription factor (TF) cone-rod homeobox (CRX) are linked with human blinding diseases that vary in their severity and age of onset. How different variants in this single TF alter its function in ways that lead to a range of phenotypes is unclear. We characterized the effects of human disease-causing variants on CRX cis-regulatory function by deploying massively parallel reporter assays (MPRAs) in mouse retina explants carrying knock-ins of two variants, one in the DNA-binding domain (p.R90W) and the other in the transcriptional effector domain (p.E168d2). The degree of reporter gene dysregulation in these mutant Crx retinas corresponds with their phenotypic severity. The two variants affect similar sets of enhancers, and p.E168d2 has distinct effects on silencers. Cis-regulatory elements (CREs) near cone photoreceptor genes are enriched for silencers that are derepressed in the presence of p.E168d2. Chromatin environments of CRX-bound loci are partially predictive of episomal MPRA activity, and distal elements whose accessibility increases later in retinal development are enriched for CREs with silencer activity. We identified a set of potentially pleiotropic regulatory elements that convert from silencers to enhancers in retinas that lack a functional CRX effector domain. Our findings show that phenotypically distinct variants in different domains of CRX have partially overlapping effects on its cis-regulatory function, leading to misregulation of similar sets of enhancers while having a qualitatively different impact on silencers.

Direct observation of a condensate effect on super-enhancer controlled gene bursting

Enhancers are distal DNA elements believed to loop and contact promoters to control gene expression. Recently, we found diffraction-sized transcriptional condensates at genes controlled by clusters of enhancers (super-enhancers). However, a direct function of endogenous condensates in controlling gene expression remains elusive. Here, we develop live-cell super-resolution and multi-color 3D-imaging approaches to investigate putative roles of endogenous condensates in the regulation of super-enhancer controlled gene Sox2. In contrast to enhancer distance, we find instead that the condensate's positional dynamics are a better predictor of gene expression. A basal gene bursting occurs when the condensate is far (>1μm), but burst size and frequency are enhanced when the condensate moves in proximity (<1μm). Perturbations of cohesin and local DNA elements do not prevent basal bursting but affect the condensate and its burst enhancement. We propose a three-way kissing model whereby the condensate interacts transiently with gene locus and regulatory DNA elements to control gene bursting.

Carl Bergmann (1814–1865) and the discovery of the anatomical site in the retina where vision is initiated

ABSTRACT A preeminent quest of nineteenth-century visual neuroscience was to identify the anatomical elements of the retina that respond to light. A major breakthrough came in 1854, when Carl Bergmann discovered through direct observation that the human fovea contains only rods and cones. On this basis, he argued that these must necessarily be the light-sensitive elements (i.e., photoreceptors) that initiate vision. Bergmann also argued that Henle’s fibers form part of the necessary anatomical link between these distal elements and the proximal ganglion cells, which transmit visual signals to the brain via the optic nerve. However, despite his achievement, Heinrich Müller, not Bergmann, is remembered as the discoverer of human photoreceptors in the literature. This article seeks to correct the record. It situates Bergmann’s work alongside that of his contemporaries, sets out his arguments and the critique he received using archival documents, and makes this history more accessible for current readers by comparing what was said to what we know now. We argue that Bergmann’s arguments are at least as compelling as those of Müller, and that he should be recognized as a codiscoverer of the anatomic site in the retina where vision is initiated.

Alternative cleavage and polyadenylation of the Ccnb1 mRNA defines accumulation of cyclin protein during the meiotic cell cycle.

Progression through the mitotic and meiotic cell cycle is driven by fluctuations in the levels of cyclins, the regulatory subunits controlling the localization and activity of CDK1 kinases. Cyclin levels are regulated through a precise balance of synthesis and degradation. Here we demonstrate that the synthesis of Cyclin B1 during the oocyte meiotic cell cycle is defined by the selective translation of mRNA variants generated through alternative cleavage and polyadenylation (APA). Using gene editing in mice, we introduced mutations into the proximal and distal polyadenylation elements of the 3' untranslated region (UTR) of the Ccnb1 mRNA. Through in vivo loss-of-function experiments, we demonstrate that the translation of mRNA with a short 3' UTR specifies Cyclin B1 protein levels that set the timing of meiotic re-entry. In contrast, translation directed by a long 3' UTR is necessary to direct Cyclin B1 protein accumulation during the MI/MII transition. These findings establish that the progression through the cell cycle is dependent on the selective translation of multiple mRNA variants generated by APA.

Asxl1-mutant Dominant Clones Have an Erythropoietic Lineage Deficiency through a Disturbed Epigenetic Landscape

Two hematologic features of red blood cells (RBCs) rank among the top three high risk factors associated with increased risk of malignant progression from clonal hematopoiesis (CH). These risk factors are macrocytosis as measured by elevated mean corpuscular volume (MCV) of 100 fL or greater and red cell distribution width (RDW) of 15% or greater. It is not known how these changes in RBC parameters are related to the leukemic transformation. Without major disturbance in the quantitative output of RBCs, these changes may reflect clone specific changes in erythropoiesis that are averaged in such measurements. We used an in vivo model of CH in zebrafish to study epigenetic and transcriptional changes in mutant clones, focusing on the erythropoietic program. In this model, mosaic somatic mutations in exon 12 of asxl1 with CRISPR-Cas9 mutagenesis are combined with color labeling of hematopoietic stem and progenitor cell (HSPC) clones. These mosaic asxl1 mutants exhibit an expansion of single-colored mutant clones to greater than 30% of hematopoietic output. No significant hematopoietic disturbance has been noted in these mutants by flow cytometric and morphologic analysis. To study clone specific effects of this epigenetic factor mutation, we sorted HSPCs from the dominant asxl1-mutant clone and compared it to HSPCs from control animals using transposase-accessible chromatin with sequencing (ATAC-seq) assay. Among over 22,000 peaks, we identified 998 (4.5%) that increased and 938 (4.2%) that decreased by 2-fold in asxl1-mutant clones compared to controls. Of the upregulated peaks, 73% were distal elements (+/- 100kb from transcriptional start sites, TSS) and 27% were proximal or promoter peaks (-2kb to +500bp from TSS), while in decreased peaks 67% were in distal elements and 33% in the proximal/promoter region. Pathway analysis of assigned genes related to decreased peaks identified a striking loss of the erythropoietic program and the heme biosynthesis process. HOMER analysis of sequences within the decreased peaks showed significant association with GATA ( q-value &amp;lt;0.001) and TRPS1 ( q-value &amp;lt;0.001) motifs. This was confirmed by individual assessment of gene loci for lineage-specifying transcriptional factors, such as tal1 and gata1a and genes in erythropoiesis, including alas2, cahz, tmod4 and the hemoglobin locus with known enhancers in nprl3 gene . Using an independently generated mutant zebrafish cohort, we performed bulk RNA-sequencing on sorted dominant clone and control HSPCs. Downregulated genes were consistent with a loss of erythroid differentiation program, with up to 16-fold downregulation of hbba1, hbaa1, alas2 and cahz although less pronounced or no significant changes were noted in erythroid transcription factors ( tal1, gata1a, lmo2) . Our data show that asxl1-mutant clones have an erythropoietic lineage defect with reduced epigenetic accessibility at lineage-specific loci with selective changes in gene expression. This ineffective erythropoiesis characterized by large MCV in mutant RBCs would increase the RDW due to the mixed contribution from mutant and wildtype clones in the peripheral blood. Our findings offer a biologic basis of the strong clinical association of abnormal RBC characteristics with higher risk CH for malignant transformation.