KDM3A and KDM3B Maintain Naïve Pluripotency Through the Regulation of Alternative Splicing.

Histone modifying enzymes play a central role in maintaining cell identity by establishing a conducive chromatin environment for lineage specific transcription factor activity. Pluripotent embryonic stem cell (ESC) identity is characterized by a lower abundance of gene repression associated histone modifications that enables rapid response to differentiation cues. The KDM3 family of histone demethylases removes the repressive histone H3 lysine 9 dimethylation (H3K9me2). Here we uncover a surprising role for the KDM3 proteins in the maintenance of the pluripotent state through post-transcriptional regulation. We find that KDM3A and KDM3B interact with RNA processing factors such as EFTUD2 and PRMT5. Acute selective degradation of the endogenous KDM3A and KDM3B proteins resulted in altered splicing independent of H3K9me2 status or catalytic activity. These splicing changes partially resemble the splicing pattern of the more blastocyst-like ground state of pluripotency and occurred in important chromatin and transcription factors such as Dnmt3b, Tbx3 and Tcf12. Our findings reveal non-canonical roles of histone demethylating enzymes in splicing to regulate cell identity.

DOT1L interaction partner AF10 controls patterning of H3K79 methylation and RNA polymerase II to maintain cell identity.

Histone 3 lysine 79 methylation (H3K79me) is enriched on gene bodies proportional to gene expression levels and serves as a strong barrier for the reprogramming of somatic cells to induced pluripotent stem cells (iPSCs). DOT1L is the sole histone methyltransferase that deposits all three orders-mono (me1), di (me2), and tri (me3) methylation-at H3K79. Here, we leverage genetic and chemical approaches to parse the specific functions of orders of H3K79me in maintaining cell identity. DOT1L interacts with AF10 (Mllt10), which recognizes unmodified H3K27 and boosts H3K79me2/3 methylation. AF10 deletion evicts H3K79me2/3 and reorganizes H3K79me1 to the transcription start site to facilitate iPSC formation in the absence of steady-state transcriptional changes. Instead, AF10 loss redistributes RNA polymerase II to a uniquely pluripotent pattern at highly expressed, rapidly transcribed housekeeping genes. Taken together, we reveal a specific mechanism for H3K79me2/3 located at the gene body in reinforcing cell identity.

DOT1L is a barrier to histone acetylation during reprogramming to pluripotency.

Embryonic stem cells (ESCs) have transcriptionally permissive chromatin enriched for gene activation-associated histone modifications. A striking exception is DOT1L-mediated H3K79 dimethylation (H3K79me2) that is considered a positive regulator of transcription. We find that ESCs are depleted for H3K79me2 at shared locations of enrichment with somatic cells, which are highly and ubiquitously expressed housekeeping genes, and have lower RNA polymerase II (RNAPII) at the transcription start site (TSS) despite greater nascent transcription. Inhibiting DOT1L increases the efficiency of reprogramming of somatic to induced pluripotent stem cells, enables an ESC-like RNAPII pattern at the TSS, and functionally compensates for enforced RNAPII pausing. DOT1L inhibition increases H3K27 methylation and RNAPII elongation-enhancing histone acetylation without changing the expression of the causal histone-modifying enzymes. Only the maintenance of elevated histone acetylation is essential for enhanced reprogramming and occurs at loci that are depleted for H3K79me2. Thus, DOT1L inhibition promotes the hyperacetylation and hypertranscription pluripotent properties.

LRRC10 regulates mammalian cardiomyocyte cell cycle during heart regeneration.

Leucine-rich repeat containing 10 (LRRC10) is a cardiomyocyte-specific protein, but its role in cardiac biology is little understood. Recently Lrrc10 was identified as required for endogenous cardiac regeneration in zebrafish; however, whether LRRC10 plays a role in mammalian heart regeneration remains unclear. In this study, we demonstrate that Lrrc10-/- knockout mice exhibit a loss of the neonatal mouse regenerative response, marked by reduced cardiomyocyte cytokinesis and increased cardiomyocyte binucleation. Interestingly, LRRC10 deletion disrupts the regenerative transcriptional landscape of the regenerating neonatal mouse heart. Remarkably, cardiac overexpression of LRRC10 restores cardiomyocyte cytokinesis, increases cardiomyocyte mononucleation, and the cardiac regenerative capacity of Lrrc10-/- mice. Our results are consistent with a model in which LRRC10 is required for cardiomyocyte cytokinesis as well as regulation of the transcriptional landscape during mammalian heart regeneration.

Inference of cell type-specific gene regulatory networks on cell lineages from single cell omic datasets.

Cell type-specific gene expression patterns are outputs of transcriptional gene regulatory networks (GRNs) that connect transcription factors and signaling proteins to target genes. Single-cell technologies such as single cell RNA-sequencing (scRNA-seq) and single cell Assay for Transposase-Accessible Chromatin using sequencing (scATAC-seq), can examine cell-type specific gene regulation at unprecedented detail. However, current approaches to infer cell type-specific GRNs are limited in their ability to integrate scRNA-seq and scATAC-seq measurements and to model network dynamics on a cell lineage. To address this challenge, we have developed single-cell Multi-Task Network Inference (scMTNI), a multi-task learning framework to infer the GRN for each cell type on a lineage from scRNA-seq and scATAC-seq data. Using simulated and real datasets, we show that scMTNI is a broadly applicable framework for linear and branching lineages that accurately infers GRN dynamics and identifies key regulators of fate transitions for diverse processes such as cellular reprogramming and differentiation.

Oct4:Sox2 binding is essential for establishing but not maintaining active and silent states of dynamically regulated genes in pluripotent cells.

Much has been learned about the mechanisms of action of pluripotency factors Oct4 and Sox2. However, as with other regulators of cell identity, little is known about the impact of disrupting their binding motifs in a native environment or the characteristics of genes they regulate. By quantitatively examining dynamic ranges of gene expression instead of focusing on conventional measures of differential expression, we found that Oct4 and Sox2 enhancer binding is strongly enriched near genes subject to large dynamic ranges of expression among cell types, with binding sites near these genes usually within superenhancers. Mutagenesis of representative Oct4:Sox2 motifs near such active, dynamically regulated genes revealed critical roles in transcriptional activation during reprogramming, with more limited roles in transcriptional maintenance in the pluripotent state. Furthermore, representative motifs near silent genes were critical for establishing but not maintaining the fully silent state, while genes whose transcript levels varied by smaller magnitudes among cell types were unaffected by nearby Oct4:Sox2 motifs. These results suggest that Oct4 and Sox2 directly establish both active and silent transcriptional states in pluripotent cells at a large number of genes subject to dynamic regulation during mammalian development, but are less important than expected for maintaining transcriptional states.

Connecting the DOTs on Cell Identity.

DOT1-Like (DOT1L) is the sole methyltransferase of histone H3K79, a modification enriched mainly on the bodies of actively transcribing genes. DOT1L has been extensively studied in leukemia were some of the most frequent onco-fusion proteins contain portions of DOT1L associated factors that mislocalize H3K79 methylation and drive oncogenesis. However, the role of DOT1L in non-transformed, developmental contexts is less clear. Here we assess the known functional roles of DOT1L both in vitro cell culture and in vivo models of mammalian development. DOT1L is evicted during the 2-cell stage when cells are totipotent and massive epigenetic and transcriptional alterations occur. Embryonic stem cell lines that are derived from the blastocyst tolerate the loss of DOT1L, while the reduction of DOT1L protein levels or its catalytic activity greatly enhances somatic cell reprogramming to induced pluripotent stem cells. DOT1L knockout mice are embryonically lethal when organogenesis commences. We catalog the rapidly increasing studies of total and lineage specific knockout model systems that show that DOT1L is broadly required for differentiation. Reduced DOT1L activity is concomitant with increased developmental potential. Contrary to what would be expected of a modification that is associated with active transcription, loss of DOT1L activity results in more upregulated than downregulated genes. DOT1L also participates in various epigenetic networks that are both cell type and developmental stage specific. Taken together, the functions of DOT1L during development are pleiotropic and involve gene regulation at the locus specific and global levels.

Dynamic regulatory module networks for inference of cell type-specific transcriptional networks.

Changes in transcriptional regulatory networks can significantly alter cell fate. To gain insight into transcriptional dynamics, several studies have profiled bulk multi-omic data sets with parallel transcriptomic and epigenomic measurements at different stages of a developmental process. However, integrating these data to infer cell type-specific regulatory networks is a major challenge. We present dynamic regulatory module networks (DRMNs), a novel approach to infer cell type-specific cis-regulatory networks and their dynamics. DRMN integrates expression, chromatin state, and accessibility to predict cis-regulators of context-specific expression, where context can be cell type, developmental stage, or time point, and uses multitask learning to capture network dynamics across linearly and hierarchically related contexts. We applied DRMNs to study regulatory network dynamics in three developmental processes, each showing different temporal relationships and measuring a different combination of regulatory genomic data sets: cellular reprogramming, liver dedifferentiation, and forward differentiation. DRMN identified known and novel regulators driving cell type-specific expression patterns, showing its broad applicability to examine dynamics of gene regulatory networks from linearly and hierarchically related multi-omic data sets.

DOT1L inhibition enhances pluripotency beyond acquisition of epithelial identity and without immediate suppression of the somatic transcriptome.

Inhibiting the histone 3 lysine 79 (H3K79) methyltransferase, disruptor of telomeric silencing 1-like (DOT1L), increases the efficiency of reprogramming somatic cells to induced pluripotent stem cells (iPSCs). Here, we find that, despite the enrichment of H3K79 methylation on thousands of actively transcribed genes in somatic cells, DOT1L inhibition (DOT1Li) does not immediately cause the shutdown of the somatic transcriptional profile to enable transition to pluripotency. Contrary to the prevalent view, DOT1Li promotes iPSC generation beyond the mesenchymal to epithelial transition and even from already epithelial cell types. DOT1Li is most potent at the midpoint of reprogramming in part by repressing Nfix that persists at late stages of reprogramming. Importantly, regulation of single genes cannot substitute for DOT1Li, demonstrating that H3K79 methylation has pleiotropic effects in maintaining cell identity.

HP1γ regulates H3K36 methylation and pluripotency in embryonic stem cells.

The heterochromatin protein 1 (HP1) family members are canonical effectors and propagators of gene repression mediated by histone H3 lysine 9 (H3K9) methylation. HP1γ exhibits an increased interaction with active transcription elongation-associated factors in embryonic stem cells (ESCs) compared to somatic cells. However, whether this association has a functional consequence remains elusive. Here we find that genic HP1γ colocalizes and enhances enrichment of transcription elongation-associated H3K36me3 rather than H3K9me3. Unexpectedly, sustained H3K36me3 deposition is dependent on HP1γ. HP1γ-deleted ESCs display reduced H3K36me3 enrichment, concomitant with decreased expression at shared genes which function to maintain cellular homeostasis. Both the H3K9me3-binding chromodomain and histone binding ability of HP1γ are dispensable for maintaining H3K36me3 levels. Instead, the chromoshadow together with the hinge domain of HP1γ that confer protein and nucleic acid-binding ability are sufficient because they retain the ability to interact with NSD1, an H3K36 methyltransferase. HP1γ-deleted ESCs have a slower self-renewal rate and an impaired ability to differentiate towards cardiac mesoderm. Our findings reveal a requirement for HP1γ in faithful establishment of transcription elongation in ESCs, which regulates pluripotency.

Methyl-Metabolite Depletion Elicits Adaptive Responses to Support Heterochromatin Stability and Epigenetic Persistence.

S-adenosylmethionine (SAM) is the methyl-donor substrate for DNA and histone methyltransferases that regulate epigenetic states and subsequent gene expression. This metabolism-epigenome link sensitizes chromatin methylation to altered SAM abundance, yet the mechanisms that allow organisms to adapt and protect epigenetic information during life-experienced fluctuations in SAM availability are unknown. We identified a robust response to SAM depletion that is highlighted by preferential cytoplasmic and nuclear mono-methylation of H3 Lys 9 (H3K9) at the expense of broad losses in histone di- and tri-methylation. Under SAM-depleted conditions, H3K9 mono-methylation preserves heterochromatin stability and supports global epigenetic persistence upon metabolic recovery. This unique chromatin response was robust across the mouse lifespan and correlated with improved metabolic health, supporting a significant role for epigenetic adaptation to SAM depletion in vivo. Together, these studies provide evidence for an adaptive response that enables epigenetic persistence to metabolic stress.

Beta Cell Dedifferentiation Induced by IRE1α Deletion Prevents Type 1 Diabetes.

Immune-mediated destruction of insulin-producing ß cells causes type 1 diabetes (T1D). However, how ß cells participate in their own destruction during the disease process is poorly understood. Here, we report that modulating the unfolded protein response (UPR) in ß cells of non-obese diabetic (NOD) mice by deleting the UPR sensor IRE1α prior to insulitis induced a transient dedifferentiation of ß cells, resulting in substantially reduced islet immune cell infiltration and ß cell apoptosis. Single-cell and whole-islet transcriptomics analyses of immature ß cells revealed remarkably diminished expression of ß cell autoantigens and MHC class I components, and upregulation of immune inhibitory markers. IRE1α-deficient mice exhibited significantly fewer cytotoxic CD8+ T cells in their pancreata, and adoptive transfer of their total T cells did not induce diabetes in Rag1-/- mice. Our results indicate that inducing ß cell dedifferentiation, prior to insulitis, allows these cells to escape immune-mediated destruction and may be used as a novel preventive strategy for T1D in high-risk individuals.

Coordinated removal of repressive epigenetic modifications during induced reversal of cell identity.

Epigenetic modifications operate in concert to maintain cell identity, yet how these interconnected networks suppress alternative cell fates remains unknown. Here, we uncover a link between the removal of repressive histone H3K9 methylation and DNA methylation during the reprogramming of somatic cells to pluripotency. The H3K9me2 demethylase, Kdm3b, transcriptionally controls DNA hydroxymethylase Tet1 expression. Unexpectedly, in the absence of Kdm3b, loci that must be DNA demethylated are trapped in an intermediate hydroxymethylated (5hmC) state and do not resolve to unmethylated cytosine. Ectopic 5hmC trapping precludes the chromatin association of master pluripotency factor, POU5F1, and pluripotent gene activation. Increased Tet1 expression is important for the later intermediates of the reprogramming process. Taken together, coordinated removal of distinct chromatin modifications appears to be an important mechanism for altering cell identity.

Defining Reprogramming Checkpoints from Single-Cell Analyses of Induced Pluripotency.

Elucidating the mechanism of reprogramming is confounded by heterogeneity due to the low efficiency and differential kinetics of obtaining induced pluripotent stem cells (iPSCs) from somatic cells. Therefore, we increased the efficiency with a combination of epigenomic modifiers and signaling molecules and profiled the transcriptomes of individual reprogramming cells. Contrary to the established temporal order, somatic gene inactivation and upregulation of cell cycle, epithelial, and early pluripotency genes can be triggered independently such that any combination of these events can occur in single cells. Sustained co-expression of Epcam, Nanog, and Sox2 with other genes is required to progress toward iPSCs. Ehf, Phlda2, and translation initiation factor Eif4a1 play functional roles in robust iPSC generation. Using regulatory network analysis, we identify a critical role for signaling inhibition by 2i in repressing somatic expression and synergy between the epigenomic modifiers ascorbic acid and a Dot1L inhibitor for pluripotency gene activation.

The role of α-ketoglutarate-dependent proteins in pluripotency acquisition and maintenance.

α-Ketoglutarate is an important metabolic intermediate that acts as a cofactor for several chromatin-modifying enzymes, including histone demethylases and the Tet family of enzymes that are involved in DNA demethylation. In this review, we focus on the function and genomic localization of these α-ketoglutarate-dependent enzymes in the maintenance of pluripotency during cellular reprogramming to induced pluripotent stem cells and in disruption of pluripotency during in vitro differentiation. The enzymatic function of many of these α-ketoglutarate-dependent proteins is required for pluripotency acquisition and maintenance. A better understanding of their specific function will be essential in furthering our knowledge of pluripotency.

Compartmentalization of HP1 Proteins in Pluripotency Acquisition and Maintenance.

The heterochromatin protein 1 (HP1) family is involved in various functions with maintenance of chromatin structure. During murine somatic cell reprogramming, we find that early depletion of HP1γ reduces the generation of induced pluripotent stem cells, while late depletion enhances the process, with a concomitant change from a centromeric to nucleoplasmic localization and elongation-associated histone H3.3 enrichment. Depletion of heterochromatin anchoring protein SENP7 increased reprogramming efficiency to a similar extent as HP1γ, indicating the importance of HP1γ release from chromatin for pluripotency acquisition. HP1γ interacted with OCT4 and DPPA4 in HP1α and HP1ß knockouts and in H3K9 methylation depleted H3K9M embryonic stem cell (ESC) lines. HP1α and HP1γ complexes in ESCs differed in association with histones, the histone chaperone CAF1 complex, and specific components of chromatin-modifying complexes such as DPY30, implying distinct functional contributions. Taken together, our results reveal the complex contribution of the HP1 proteins to pluripotency.

Primary Cilium-Mediated Retinal Pigment Epithelium Maturation Is Disrupted in Ciliopathy Patient Cells.

Primary cilia are sensory organelles that protrude from the cell membrane. Defects in the primary cilium cause ciliopathy disorders, with retinal degeneration as a prominent phenotype. Here, we demonstrate that the retinal pigment epithelium (RPE), essential for photoreceptor development and function, requires a functional primary cilium for complete maturation and that RPE maturation defects in ciliopathies precede photoreceptor degeneration. Pharmacologically enhanced ciliogenesis in wild-type induced pluripotent stem cells (iPSC)-RPE leads to fully mature and functional cells. In contrast, ciliopathy patient-derived iPSC-RPE and iPSC-RPE with a knockdown of ciliary-trafficking protein remain immature, with defective apical processes, reduced functionality, and reduced adult-specific gene expression. Proteins of the primary cilium regulate RPE maturation by simultaneously suppressing canonical WNT and activating PKCδ pathways. A similar cilium-dependent maturation pathway exists in lung epithelium. Our results provide insights into ciliopathy-induced retinal degeneration, demonstrate a developmental role for primary cilia in epithelial maturation, and provide a method to mature iPSC epithelial cells for clinical applications.

Elucidating Protein-DNA Interactions in Human Alphoid Chromatin via Hybridization Capture and Mass Spectrometry.

The centromere is the chromosomal locus where the kinetochore forms and is critical for ensuring proper segregation of sister chromatids during cell division. A substantial amount of effort has been devoted to understanding the characteristic features and roles of the centromere, yet some fundamental aspects of the centromere, such as the complete list of elements that define it, remain obscure. It is well-known that human centromeres include a highly repetitive class of DNA known as alpha satellite, or alphoid, DNA. We present here the first DNA-centric examination of human protein-alpha satellite interactions, employing an approach known as HyCCAPP (hybridization capture of chromatin-associated proteins for proteomics) to identify the protein components of alphoid chromatin in a human cell line. Using HyCCAPP, cross-linked alpha satellite chromatin was isolated from cell lysate, and captured proteins were analyzed via mass spectrometry. After being compared to proteins identified in control pulldown experiments, 90 proteins were identified as enriched at alphoid DNA. This list included many known centromere-binding proteins in addition to multiple novel alpha satellite-binding proteins, such as LRIF1, a heterochromatin-associated protein. The ability of HyCCAPP to reveal both known as well as novel alphoid DNA-interacting proteins highlights the validity and utility of this approach.

Chromatin module inference on cellular trajectories identifies key transition points and poised epigenetic states in diverse developmental processes.

Changes in chromatin state play important roles in cell fate transitions. Current computational approaches to analyze chromatin modifications across multiple cell types do not model how the cell types are related on a lineage or over time. To overcome this limitation, we developed a method called Chromatin Module INference on Trees (CMINT), a probabilistic clustering approach to systematically capture chromatin state dynamics across multiple cell types. Compared to existing approaches, CMINT can handle complex lineage topologies, capture higher quality clusters, and reliably detect chromatin transitions between cell types. We applied CMINT to gain novel insights in two complex processes: reprogramming to induced pluripotent stem cells (iPSCs) and hematopoiesis. In reprogramming, chromatin changes could occur without large gene expression changes, different combinations of activating marks were associated with specific reprogramming factors, there was an order of acquisition of chromatin marks at pluripotency loci, and multivalent states (comprising previously undetermined combinations of activating and repressive histone modifications) were enriched for CTCF. In the hematopoietic system, we defined critical decision points in the lineage tree, identified regulatory elements that were enriched in cell-type-specific regions, and found that the underlying chromatin state was achieved by specific erasure of preexisting chromatin marks in the precursor cell or by de novo assembly. Our method provides a systematic approach to model the dynamics of chromatin state to provide novel insights into the relationships among cell types in diverse cell-fate specification processes.

Alternative Routes to Induced Pluripotent Stem Cells Revealed by Reprogramming of the Neural Lineage.

During the reprogramming of mouse embryonic fibroblasts (MEFs) to induced pluripotent stem cells, the activation of pluripotency genes such as NANOG occurs after the mesenchymal to epithelial transition. Here we report that both adult stem cells (neural stem cells) and differentiated cells (astrocytes) of the neural lineage can activate NANOG in the absence of cadherin expression during reprogramming. Gene expression analysis revealed that only the NANOG+E-cadherin+ populations expressed stabilization markers, had upregulated several cell cycle genes; and were transgene independent. Inhibition of DOT1L activity enhanced both the numbers of NANOG+ and NANOG+E-cadherin+ colonies in neural stem cells. Expressing SOX2 in MEFs prior to reprogramming did not alter the ratio of NANOG colonies that express E-cadherin. Taken together these results provide a unique pathway for reprogramming taken by cells of the neural lineage.