Mouse ES Research Articles

The 129 strain-derived passenger mutations in ACKR1-deficient mice alter the expression of PYHIN and Fc-gamma receptor genes.

A majority of genetically modified mice have been produced using 129 strain-derived embryonic stem cells (ESCs). Despite ample backcrosses with other strains, these may retain characteristic for 129 passenger mutations leading to confounding phenotypes unrelated to targeted genes. Here we show that widely used Ackr1-/-129ES mice have approximately 6Mb of the 129-derived genome retained adjacently to the Ackr1 locus on chromosome 1, including several characteristic polymorphisms. These most notably affect the expression of PYHIN and Fc-gamma receptor genes in myeloid cells resulting in the overproduction of IL-1β by activated macrophages and the loss of Fc-gamma receptors on myeloid progenitor cells. Therefore, caution is warranted when interpreting Ackr1-/-129ES mouse phenotypes as being solely due to the ACKR1 deficiency. Our findings call for a careful reevaluation of data from previous studies using Ackr1-/-129ES mice and underscore the limitations and pitfalls inherent to mouse models produced using traditional genetic engineering techniques involving 129 ESCs.

Enhancer regulation by H3K4me1 methyltransferases MLL3/MLL4

Enhancers control cell type‐specific gene expression and are marked by H3K4me1. Active enhancers are further marked by H3K27ac. We identified MLL3/MLL4 as major H3K4me1 methyltransferases and CBP/p300 as the H3K27 acetyltransferases in mammalian cells (EMBO J 2011; eLife 2013). During differentiation of adipocytes, myocytes and ES cells, MLL3/MLL4 co‐localize with lineage‐determining transcription factors (LDTFs), CBP/p300, Brd4 and MED1 on active enhancers. MLL3/MLL4 are required for enhancer activation, enhancer‐promoter interaction, cell type‐specific gene expression, and cell differentiation (eLife 2013; PNAS 2016; Nat Comm 2017). MLL3/MLL4 control cell differentiation by orchestrating CBP/p300‐mediated enhancer activation (NAR 2017). Ectopic expression of H3.3K4M, an inhibitor of H3K4 methylation, or deletion of the enzymatic SET domain, destabilizes MLL3/MLL4 proteins, prevents enhancer activation, and impairs cell differentiation and tissue development (NAR 2019).Using proteomic approaches, we found that endogenous MLL4 complex associates with SWI/SNF chromatin remodeling complex components in cells. Using adipogenesis as a model system, we establish an interdependent relationship between SWI/SNF complex BAF and MLL4 in promoting cell type‐specific enhancer activation by LDTFs, which rectifies seemingly conflicting results from previous studies (Nat Comm 2021).We showed that MLL4 protein, rather than MLL4‐mediated H3K4me1, controls p300 recruitment to enhancers during ES cell differentiation, suggesting that MLL4 regulates enhancer activation independent of H3K4me1 (PNAS 2016). We have generated enzyme‐dead MLL3/MLL4 double knockin ES cells and mice by CRISPR. Initial analyses indicate that MLL3/MLL4 regulate ES cell differentiation and mouse embryonic development through both enzymatic activity‐dependent and ‐independent mechanisms.

Synthetic modified Fezf2 mRNA (modRNA) with concurrent small molecule SIRT1 inhibition enhances refinement of cortical subcerebral/corticospinal neuron identity from mouse embryonic stem cells.

During late embryonic development of the cerebral cortex, the major class of cortical output neurons termed subcerebral projection neurons (SCPN; including the predominant population of corticospinal neurons, CSN) and the class of interhemispheric callosal projection neurons (CPN) initially express overlapping molecular controls that later undergo subtype-specific refinements. Such molecular refinements are largely absent in heterogeneous, maturation-stalled, neocortical-like neurons (termed “cortical” here) spontaneously generated by established embryonic stem cell (ES) and induced pluripotent stem cell (iPSC) differentiation. Building on recently identified central molecular controls over SCPN development, we used a combination of synthetic modified mRNA (modRNA) for Fezf2, the central transcription factor controlling SCPN specification, and small molecule screening to investigate whether distinct chromatin modifiers might complement Fezf2 functions to promote SCPN-specific differentiation by mouse ES (mES)-derived cortical-like neurons. We find that the inhibition of a specific histone deacetylase, Sirtuin 1 (SIRT1), enhances refinement of SCPN subtype molecular identity by both mES-derived cortical-like neurons and primary dissociated E12.5 mouse cortical neurons. In vivo, we identify that SIRT1 is specifically expressed by CPN, but not SCPN, during late embryonic and postnatal differentiation. Together, these data indicate that SIRT1 has neuronal subtype-specific expression in the mouse cortex in vivo, and that its inhibition enhances subtype-specific differentiation of highly clinically relevant SCPN / CSN cortical neurons in vitro.

1155 Are common processes involved in both implanting an embryo and ovarian cancer?

Implantation is fundamental to successful establishment of both the trophoblast (placenta) and embryo. However, there are practical, ethical and legal restrictions to studying this process in humans. Implantation of free-floating cells originating from the oviduct leads to benign ectopic lesions (endosalpingiosis, ES) and may also play a key role in the pathogenesis of gynecologic malignancies. We hypothesized that by understanding the mechanisms driving ES, we would gain crucial insight into early pregnancy implantation and failures. We aimed to develop a mouse model to interrogate the role of implantation signaling factors in promoting benign ectopic lesions such as ES, as a novel model for early pregnancy and ovarian cancer. We developed an animal model of ES using auto-fluorescing (inherent in the donor mouse cells) oviductal tissue or oviductal epithelia cells (OECs). Utilizing this ES model, donor mice were treated with hormones to induce infertility by blocking implantation or with a vehicle control (Fig. 1). Oviductal tissue from these mice was harvested and implanted into the peritoneal cavity of recipient wild type mice. Recipient mice are monitored for a month before mice are imaged for the formation of auto-fluorescing legions resulting from the donor tissue. We observed 100% successful implantation rate in our ES mouse model of oviductal tissue was administered (Fig. 2). When isolated OECs were used, they required the co-injection of endometrium to successfully implant, however only 30% successfully implanted. We successfully created a model of ES that allows us to identify growth of lesions in vivo allowing us to study the mechanisms of implantation. We anticipate that by blocking key signals of implantation the formation of oviductal lesions in the peritoneal cavity will be blocked or significantly reduced. Success with this model will allow us to not only understand ES but has applications to the molecular mechanisms of pregnancy success and failures, alongside ovarian cancers derived from oviduct and endometrial cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Kyoto probe-1 reveals phenotypic differences between mouse ES cells and iTS-P cells

Kyoto probe 1 (KP-1) rapidly distinguishes between human ES/iPS (hES/iPS) cells and their differentiated cells. Recently, we generated induced tissue-specific stem cells from pancreas (iTS-P cells) using reprogramming factors and tissue-specific selection. The iTS-P cells have self-renewal potential, and subcutaneously transplanting them into immunodeficient mice did not generate teratomas. In this study, we applied KP-1 to analyze mouse ES (mES) cells and mouse iTS-P (miTS-P) cells. KP-1 completely stained mES cells in colonies, but only miTS-P cells at the edge of a colony. This difference was caused by cell type-specific expression of different ABC transporters. These finding suggest that KP-1 will be useful for distinguishing between iPS and iTS-P cells.

Sirt6 regulates efficiency of mouse somatic reprogramming and maintenance of pluripotency

BackgroundMouse somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by defined factors known to regulate pluripotency, including Oct4, Sox2, Klf4, and c-Myc. It has been reported that Sirtuin 6 (Sirt6), a member of the sirtuin family of NAD+-dependent protein deacetylases, is involved in embryonic stem cell differentiation. However, whether and how Sirt6 influences epigenetic reprogramming remains unknown.MethodsMouse embryonic fibroblasts isolated from transgenic Oct4-GFP reporter mice with or without Sirt6 were used for reprogramming by Yamanaka factors. Alkaline phosphatase-positive and OCT4-GFP-positive colony were counted to calculate reprogramming efficiency. OP9 feeder cell co-culture system was used to measure the hematopoietic differentiation from mouse ES and iPS cells. RNA sequencing was measured to identify the differential expressed genes due to loss of Sirt6 in somatic and pluripotent cells.ResultsIn this study, we provide evidence that Sirt6 is involved in mouse somatic reprogramming. We found that loss of function of Sirt6 could significantly decrease reprogramming efficiency. Furthermore, we showed that Sirt6-null iPS-like cell line has intrinsically a differentiation defect even though the establishment of normal self-renewal. Particularly, by performing transcriptome analysis, we observed that several pluripotent transcriptional factors increase in knockout cell line, which explains the underlying loss of pluripotency in Sirt6-null iPS-like cell line.ConclusionsTaken together, we have identified a new regulatory role of Sirt6 in reprogramming and maintenance of pluripotency.

Hoxa1 targets signaling pathways during neural differentiation of ES cells and mouse embryogenesis

Hoxa1 has important functional roles in neural crest specification, hindbrain patterning and heart and ear development, yet the enhancers and genes that are targeted by Hoxa1 are largely unknown. In this study, we performed a comprehensive analysis of Hoxa1 target genes using genome-wide Hoxa1 binding data in mouse ES cells differentiated with retinoic acid (RA) into neural fates in combination with differential gene expression analysis in Hoxa1 gain- and loss-of-function mouse and zebrafish embryos. Our analyses reveal that Hoxa1-bound regions show epigenetic marks of enhancers, occupancy of Hox cofactors and differential expression of nearby genes, suggesting that these regions are enriched for enhancers. In support of this, 80 of them mapped to regions with known reporter activity in transgenic mouse embryos based on the Vista enhancer database. Two additional enhancers in Dok5 and Wls1 were shown to mediate neural expression in developing mouse and zebrafish. Overall, our analysis of the putative target genes indicate that Hoxa1 has input to components of major signaling pathways, including Wnt, TGF-β, Hedgehog and Hippo, and frequently does so by targeting multiple components of a pathway such as secreted inhibitors, ligands, receptors and down-stream components. We also identified genes implicated in heart and ear development, neural crest migration and neuronal patterning and differentiation, which may underlie major Hoxa1 mutant phenotypes. Finally, we found evidence for a high degree of evolutionary conservation of many binding regions and downstream targets of Hoxa1 between mouse and zebrafish. Our genome-wide analyses in ES cells suggests that we have enriched for in vivo relevant target genes and pathways associated with functional roles of Hoxa1 in mouse development.

Chromatin priming of genes in development: Concepts, mechanisms and consequences.

During ontogeny, cells progress through multiple alternate differentiation states by activating distinct gene regulatory networks. In this review, we highlight the important role of chromatin priming in facilitating gene activation during lineage specification and in maintaining an epigenetic memory of previous gene activation. We show that chromatin priming is part of a hugely diverse repertoire of regulatory mechanisms that genes use to ensure that they are expressed at the correct time, in the correct cell type, and at the correct level, but also that they react to signals. We also emphasize how increasing our knowledge of these principles could inform our understanding of developmental failure and disease.

Production of stable GFP-expressing neural cells from P19 embryonal carcinoma stem cells

Murine P19 embryonal carcinoma (EC) cells are convenient to differentiate into all germ layer derivatives. One of the advantages of P19 cells is that the exogenous DNA can be easily inserted into them. Here, at the first part of this study, we generated stable GFP-expressing P19 cells (P19-GFP+). FACS and western-blot analysis confirmed stable expression of GFP in the cells. We previously demonstrated the efficient induction of neuronal differentiation from mouse ES and EC cells by application of a neuroprotective drug, selegiline In the second part of this study selegiline was used to induce differentiation of P19-GFP+ into stable GFP-expressing neuron-like cells. Cresyl violet staining confirmed neuronal morphology of the differentiated cells. Furthermore, real-time PCR and immunoflourescence approved the expression of neuron specific markers. P19-GFP+ cells were able to survive, migrate and integrated into host tissues when transplanted to developing chick embryo CNS. The obtained live GFP-expressing cells can be used as an abundant source of developmentally pluripotent material for transplantation studies, investigating the cellular and molecular aspects of early differentiation.

Abstract 65: Latrophilin-2 is a Specific Cell-surface Marker for Cardiac Progenitor Cells and Specifies Cardiac Lineage Commitment and Development

Backgrounds: The identification of a lineage-specific marker plays a pivotal role in understanding developmental process and is utilized to isolate a certain cell type with high purity for the therapeutic purpose. We here report a new cardiac-specific marker, and demonstrate its functional significance in the cardiac development. Methods and Results: When mouse pluripotent stem cells (ES and iPS cells) were stimulated with BMP4, Activin A, bFGF and VEGF, they differentiated into cardiac cells. To screen cell-surface expressing molecules on cardiac progenitor cells compared to undifferentiated mouse iPS and ES cells, we isolated Flk1+/PDGFRa+ cells at differentiation day 4 and performed microarray analysis. Among candidates, we identified a new G protein-coupled receptor, Latrophilin-2 (LPHN2) whose signaling pathway and its effect on cardiac differentiation is unknown. In sorting experiments under cardiac differentiation condition, LPHN2+ cells derived from pluripotent stem cells strongly expressed cardiac-related genes (Mesp1, Nkx2.5, aMHC and cTnT) and exclusively gave rise to beating cardiomyocytes, as compared with LPHN2- cells. LPHN2-/- mice revealed embryonically lethal and huge defects in cardiac development. Interestingly, LPHN2+/- heterozygotes were alive and fertile. For the purpose of cardiac regeneration, we transplanted iPS-derived LPHN2+ cells into the infarcted heart of adult mice. LPHN2+ cells differentiated into cardiomyocytes, and systolic function of left ventricle was improved and infarct size was reduced. We confirmed LPHN2 expression on human iPS and ES cell-derived cardiac progenitor cells and human heart. Conclusions: We demonstrate that LPHN2 is a functionally significant and cell-surface expressing marker for both mouse and human cardiac progenitor and cardiomyocytes. Our findings provide a valuable tool for isolating cardiac lineage cells from pluripotent stem cells and an insight into cardiac development and regeneration.

Making gametes from pluripotent stem cells--a promising role for very small embryonic-like stem cells.

The urge to have one’s own biological child supersedes any desire in life. Several options have been used to obtain gametes including pluripotent stem cells (embryonic ES and induced pluripotent iPS stem cells); gonadal stem cells (spermatogonial SSCs, ovarian OSCs stem cells), bone marrow, mesenchymal cells and fetal skin. However, the field poses a huge challenge including inefficient existing protocols for differentiation, epigenetic and genetic changes associated with extensive in vitro manipulation and also ethical/regulatory constraints. A tremendous leap in the field occurred using mouse ES and iPS cells wherein they were first differentiated into epiblast-like cells and then primordial germ cell-like cells. These on further development produced sperm, oocytes and live offspring (had associated genetic problems). Evidently differentiating pluripotent stem cells into primordial germ cells (PGCs) remains a major bottleneck. Against this backdrop, we propose that a novel population of pluripotent stem cells termed very small embryonic-like stem cells (VSELs) may serve as an alternative, potential source of autologus gametes, keeping in mind that they are indeed PGCs surviving in adult mammalian ovaries and testes. Both VSELs and PGCs are pluripotent, relatively quiescent because of epigenetic modifications of parentally imprinted genes loci like Igf2-H19 and KCNQ1p57, share several markers like Stella, Fragilis, Mvh, Dppa2, Dppa4, Sall4, Blimp1 and functional receptors. VSELs are localized in the basement membrane of seminiferous tubules in testis and in the ovary surface epithelium. Ovarian stem cells from mouse, rabbit, sheep, marmoset and humans (menopausal women and those with premature ovarian failure) spontaneously differentiate into oocyte-like structures in vitro with no additional requirement of growth factors. Thus a more pragmatic option to obtain autologus gametes may be the pluripotent VSELs and if we could manipulate them in vivo – existing ethical and epigenetic/genetic concerns associated with in vitro culture may also be minimized. The field of oncofertility may undergo a sea-change and existing strategies of cryopreservation of gametes and gonadal tissue for fertility preservation in cancer patients will necessitate a revision. However, first the scientific community needs to arrive at a consensus about VSELs in the gonads and then work towards exploiting their potential.

Point Mutation in Syntaxin-1A Causes Abnormal Vesicle Recycling, Behaviors, and Short Term Plasticity

Syntaxin-1A is a t-SNARE that is involved in vesicle docking and vesicle fusion; it is important in presynaptic exocytosis in neurons because it interacts with many regulatory proteins. Previously, we found the following: 1) that autophosphorylated Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), an important modulator of neural plasticity, interacts with syntaxin-1A to regulate exocytosis, and 2) that a syntaxin missense mutation (R151G) attenuated this interaction. To determine more precisely the physiological importance of this interaction between CaMKII and syntaxin, we generated mice with a knock-in (KI) syntaxin-1A (R151G) mutation. Complexin is a molecular clamp involved in exocytosis, and in the KI mice, recruitment of complexin to the SNARE complex was reduced because of an abnormal CaMKII/syntaxin interaction. Nevertheless, SNARE complex formation was not inhibited, and consequently, basal neurotransmission was normal. However, the KI mice did exhibit more enhanced presynaptic plasticity than wild-type littermates; this enhanced plasticity could be associated with synaptic response than did wild-type littermates; this pronounced response included several behavioral abnormalities. Notably, the R151G phenotypes were generally similar to previously reported CaMKII mutant phenotypes. Additionally, synaptic recycling in these KI mice was delayed, and the density of synaptic vesicles was reduced. Taken together, our results indicated that this single point mutation in syntaxin-1A causes abnormal regulation of neuronal plasticity and vesicle recycling and that the affected syntaxin-1A/CaMKII interaction is essential for normal brain and synaptic functions in vivo.

A prominent and conserved role for YY1 in Xist transcriptional activation

Dosage compensation for X-linked genes in female mammals relies on X-chromosome inactivation. This process involves monoallelic up-regulation of the non-coding Xist RNA which coats in cis the chromosome and triggers epigenetic reprogramming that will prevent chromosome-wide transcription. Xist transcription is controlled, directly and/or indirectly, by several ncRNA (Tsix, Jpx, Ftx) and factors (Sox2, Oct4, Nanog, Rex1, Rnf12). However, mechanisms leading to Xist monoallelic regulation remain poorly understood. Using mouse ES and differentiating cells, we identified specific YY1 and CTCF interacting sites on the Xist locus of the inactive X-chromosome. We show that this monoallelic binding is controlled by DNA methylation. To gain further insights into YY1 functional role on the Xist locus, we conducted YY1 knockdown experiments. We show by RNA-FISH that depletion of YY1 in female somatic cells impairs the accumulation of Xist on the inactive X-chromosome. This is likely due to a transcriptional effect as we observe a drastic reduction of both spliced and unspliced Xist RNA levels. This hypothesis is further reinforced by the in vitro analysis Xist promoter activity, which displays strict dependency on the YY1 binding sites. Importantly, YY1 is also necessary for the upregulation of Xist that triggers X-chromosome inactivation. Taken together, these results suggest a strong requirement for YY1 in the upregulation and maintenance of Xist transcription. Importantly, we demonstrate that the function of YY1 in the control of Xist expression is conserved in humans and predicted in other mammalian species. These results highlight the importance of YY1 both in the monoallelic upregulation of Xist at the exit of pluripotency and in the maintenance of its expression in somatic cells. Taken together with previous studies, we propose that through its dual action on Tsix and Xist, YY1 acts as a bimodal transcriptional regulator of X-inactivation.

Genome-wide comparison of DNA hydroxymethylation in mouse embryonic stem cells and neural progenitor cells by a new comparative hMeDIP-seq method

The genome-wide distribution patterns of the ‘6th base’ 5-hydroxymethylcytosine (5hmC) in many tissues and cells have recently been revealed by hydroxymethylated DNA immunoprecipitation (hMeDIP) followed by high throughput sequencing or tiling arrays. However, it has been challenging to directly compare different data sets and samples using data generated by this method. Here, we report a new comparative hMeDIP-seq method, which involves barcoding different input DNA samples at the start and then performing hMeDIP-seq for multiple samples in one hMeDIP reaction. This approach extends the barcode technology from simply multiplexing the DNA deep sequencing outcome and provides significant advantages for quantitative control of all experimental steps, from unbiased hMeDIP to deep sequencing data analysis. Using this improved method, we profiled and compared the DNA hydroxymethylomes of mouse ES cells (ESCs) and mouse ESC-derived neural progenitor cells (NPCs). We identified differentially hydroxymethylated regions (DHMRs) between ESCs and NPCs and uncovered an intricate relationship between the alteration of DNA hydroxymethylation and changes in gene expression during neural lineage commitment of ESCs. Presumably, the DHMRs between ESCs and NPCs uncovered by this approach may provide new insight into the function of 5hmC in gene regulation and neural differentiation. Thus, this newly developed comparative hMeDIP-seq method provides a cost-effective and user-friendly strategy for direct genome-wide comparison of DNA hydroxymethylation across multiple samples, lending significant biological, physiological and clinical implications.

Signaling axis involving Hedgehog, Notch, and Scl promotes the embryonic endothelial-to-hematopoietic transition

During development, the hematopoietic lineage transits through hemogenic endothelium, but the signaling pathways effecting this transition are incompletely characterized. Although the Hedgehog (Hh) pathway is hypothesized to play a role in patterning blood formation, early embryonic lethality of mice lacking Hh signaling precludes such analysis. To determine a role for Hh signaling in patterning of hemogenic endothelium, we assessed the effect of altered Hh signaling in differentiating mouse ES cells, cultured mouse embryos, and developing zebrafish embryos. In differentiating mouse ES cells and mouse yolk sac cultures, addition of Indian Hh ligand increased hematopoietic progenitors, whereas chemical inhibition of Hh signaling reduced hematopoietic progenitors without affecting primitive streak mesoderm formation. In the setting of Hh inhibition, induction of either Notch signaling or overexpression of Stem cell leukemia (Scl)/T-cell acute lymphocytic leukemia protein 1 rescued hemogenic vascular-endothelial cadherin(+) cells and hematopoietic progenitor formation. Together, our results reveal that Scl overexpression is sufficient to rescue the developmental defects caused by blocking the Hh and Notch pathways, and inform our understanding of the embryonic endothelial-to-hematopoietic transition.

Lmx1a regulates dopamine transporter gene expression during ES cell differentiation and mouse embryonic development

Midbrain dopaminergic neurons are implicated in various neurological and psychiatric diseases as well as drug addiction. Thus, the study of their generation and maintenance is pivotal to further our understanding of these disease-underlying mechanisms and development of novel therapeutics. Here, using an embryonic stem cell in vitro differentiation system and mutant dreher mouse, we showed that Lmx1a, an early regulator of midbrain dopamine neural progenitor phenotype specification, is also involved in the regulation of midbrain dopaminergic maturation by regulating gene expression of the dopamine transporter. Forced expression of Lmx1a induced dopamine transporter expression precociously in immature dopaminergic neurons, accompanied by significant increase in specific dopamine uptake. Lmx1a binds to well-conserved sequences in the dopamine transporter promoter region, and this binding sequence directs Lmx1a-dependent activation of reporter gene expression. Furthermore, during mouse embryonic development, dopamine transporter was more severely affected by Lmx1a mutation compared to other dopamine markers such as tyrosine hydroxylase and dopa decarboxylase, again supporting the role of Lmx1a in midbrain dopaminergic maturation in vivo. Thus, this study demonstrates that dopamine transporter is a direct target of Lmx1a and emphasizes a novel role of Lmx1a as one of regulators of mature midbrain dopaminergic neurotransmitter phenotypes.

Generation of mouse ES cell lines engineered for the forced induction of transcription factors

Here we report the generation and characterization of 84 mouse ES cell lines with doxycycline-controllable transcription factors (TFs) which, together with the previous 53 lines, cover 7–10% of all TFs encoded in the mouse genome. Global gene expression profiles of all 137 lines after the induction of TFs for 48 hrs can associate each TF with the direction of ES cell differentiation, regulatory pathways, and mouse phenotypes. These cell lines and microarray data provide building blocks for a variety of future biomedical research applications as a community resource.

Role of hSET1 Complex in Epigenetic Controls of HoxB4 Expression and Development of Hematopoietic Stem Cells

Abstract 212Hox genes play an important role in embryonic development and are sequentially activated in a temporal and spatial fashion. Among them, HoxB4 regulates the self-renewal ability of adult and embryonic hematopoietic stem cells. It was shown that USF proteins positively regulate the transcription of HoxB4 gene. However, the epigenetic mechanism specifically controlling HoxB4 transcription during HSC formation remains unknown. In this report, we found that USF1 interacts with histone H3K4 methylatransferase SET1 complex, but not MLL proteins. The interaction is mediated by SET1/MLL core component, ASH2L. Both USF1 and hSET1 are colocalized at the HoxB4 promoter in hematopoietic progenitors, which is accompanied by H3K4 trimethylation and transcriptional activation. siRNA-mediated knockdown of both SET1 and HCF1 specifically impaired H3K4 trimetylation and the HoxB4 promoter-driven reporter gene activity. Overexpression of AUSF1, a dominant negative mutant of USF1 in which the basic DNA recognition domain is deleted and thereby, inhibits endogenous USF DNA binding activity, leads to dramatically decrease in HoxB4 transcription, disruption of embryonic body formation and inhibition of hematopoietic stem cell population in mouse ES (mES) cells and the AUSF1 transgenic mice. The suppression of USF activity drastically reduces SET1 recruitment, H3K4 trimethylation, and pol II binding at the HoxB4 promoter. Furthermore, knock-down of SET1, an enzymatic subunit of the SET1 complex, also resulted in reduction of HoxB4 transcription and impairment of hematopoietic differentiation capacity of mES cells that are accompanied by reducing the HoxB4 promoter H3K4 methylation and recruitment of RNA PolII machinery. Thus, our data reveal that recruitment of hSET1 complex and its H3K4 methyltransferase activity by USF1 is essential for HoxB4 transcription and expansion of hematopoietic stem cells. Disclosures:No relevant conflicts of interest to declare.

Expression regulation of GAD forms in mouse ES and P19 cells

Expression regulation of GAD forms in mouse ES and P19 cells

Directed Differentiation of Human Embryonic Stem Cells to Interrogate the Cardiac Gene Regulatory Network

The limited ability of the heart to regenerate has prompted development of new systems to produce cardiomyocytes for therapeutics. While differentiation of human embryonic stem cells (hESCs) into cardiomyocytes has been well documented, the process remains inefficient and/or expensive, and progress would be facilitated by better understanding the early genetic events that cause cardiac specification. By maintaining a transgenic cardiac-specific MYH6-monomeric red fluorescent protein (mRFP) reporter hESC line in conditions that promote pluripotency, we tested the ability of combinations of 15 genes to induce cardiac specification. Screening identified GATA4 plus TBX5 as the minimum requirement to activate the cardiac gene regulatory network and produce mRFP(+) cells, while a combination of GATA4, TBX5, NKX2.5, and BAF60c (GTNB) was necessary to generate beating cardiomyocytes positive for cTnI and α-actinin. Including the chemotherapeutic agent, Ara-C, from day 10 of induced differentiation enriched for cTnI/α-actinin double positive cells to 45%. Transient expression of GTNB for 5-7 days was necessary to activate the cardiogenesis through progenitor intermediates in a manner consistent with normal heart development. This system provides a route to test the effect of different factors on human cardiac differentiation and will be useful in understanding the network failures that underlie disease phenotypes.