ASXL2 regulates hematopoiesis in mice and its deficiency promotes myeloid expansion.

Chromosomal translocation t(8;21)(q22;q22) which leads to the generation of oncogenic RUNX1-RUNX1T1 (AML1-ETO) fusion is observed in approximately 10% of acute myelogenous leukemia (AML). To identify somatic mutations that co-operate with t(8;21)-driven leukemia, we performed whole and targeted exome sequencing of an Asian cohort at diagnosis and relapse. We identified high frequency of truncating alterations in ASXL2 along with recurrent mutations of KIT, TET2, MGA, FLT3, and DHX15 in this subtype of AML. To investigate in depth the role of ASXL2 in normal hematopoiesis, we utilized a mouse model of ASXL2 deficiency. Loss of ASXL2 caused progressive hematopoietic defects characterized by myeloid hyperplasia, splenomegaly, extramedullary hematopoiesis, and poor reconstitution ability in transplantation models. Parallel analyses of young and >1-year old Asxl2-deficient mice revealed age-dependent perturbations affecting, not only myeloid and erythroid differentiation, but also maturation of lymphoid cells. Overall, these findings establish a critical role for ASXL2 in maintaining steady state hematopoiesis, and provide insights into how its loss primes the expansion of myeloid cells.

Proteomic characterization of epicardial-myocardial signaling reveals novel regulatory networks including a role for NF-κB in epicardial EMT.

Signaling between the epicardium and underlying myocardium is crucial for proper heart development. The complex molecular interactions and regulatory networks involved in this communication are not well understood. In this study, we integrated mass spectrometry with bioinformatics to systematically characterize the secretome of embryonic chicken EPDC-heart explant (EHE) co-cultures. The 150-protein secretome dataset established greatly expands the knowledge base of the molecular players involved in epicardial-myocardial signaling. We identified proteins and pathways that are implicated in epicardial-myocardial signaling for the first time, as well as new components of pathways that are known to regulate the crosstalk between epicardium and myocardium. The large size of the dataset enabled bioinformatics analysis to deduce networks for the regulation of specific biological processes and predicted signal transduction nodes within the networks. We performed functional analysis on one of the predicted nodes, NF-κB, and demonstrate that NF-κB activation is an essential step in TGFß2/PDGFBB-induced cardiac epithelial-to-mesenchymal transition. In summary, we have generated a global perspective of epicardial-myocardial signaling for the first time, and our findings open exciting new avenues for investigating the molecular basis of heart development and regeneration.

Asxl2-/- Mice Exhibit De Novo Cardiomyocyte Production during Adulthood.

Heart attacks affect more than seven million people worldwide each year. A heart attack, or myocardial infarction, may result in the death of a billion cardiomyocytes within hours. The adult mammalian heart does not have an effective mechanism to replace lost cardiomyocytes. Instead, lost muscle is replaced with scar tissue, which decreases blood pumping ability and leads to heart failure over time. Here, we report that the loss of the chromatin factor ASXL2 results in spontaneous proliferation and cardiogenic differentiation of a subset of interstitial non-cardiomyocytes. The adult Asxl2-/- heart displays spontaneous overgrowth without cardiomyocyte hypertrophy. Thymidine analog labeling and Ki67 staining of 12-week-old hearts revealed 3- and 5-fold increases of proliferation rate for vimentin⁺ non-cardiomyocytes in Asxl2-/- over age- and sex-matched wildtype controls, respectively. Approximately 10% of proliferating non-cardiomyocytes in the Asxl2-/- heart express the cardiogenic marker NKX2-5, a frequency that is ~7-fold higher than that observed in the wildtype. EdU lineage tracing experiments showed that ~6% of pulsed-labeled non-cardiomyocytes in Asxl2-/- hearts differentiate into mature cardiomyocytes after a four-week chase, a phenomenon not observed for similarly pulse-chased wildtype controls. Taken together, these data indicate de novo cardiomyocyte production in the Asxl2-/- heart due to activation of a population of proliferative cardiogenic non-cardiomyocytes. Our study suggests the existence of an epigenetic barrier to cardiogenicity in the adult heart and raises the intriguing possibility of unlocking regenerative potential via transient modulation of epigenetic activity.

WTIP interacts with ASXL2 and blocks ASXL2-mediated activation of retinoic acid signaling.

The Asx-like (ASXL) family proteins are chromatin factors that play dual roles in transcriptional activation and repression. ASXL2 is highly expressed in the heart and is required for proper heart development and function. Here, we identify a novel ASXL2-binding partner, the LIM domain-containing protein WTIP. Genetic and biochemical assays show a direct interaction between ASXL2 and WTIP. In HeLa cells, ASXL2 enhances retinoic acid-dependent luciferase activity, while WTIP represses it. Furthermore, WTIP blocks ASXL2's stimulatory effect on transcription. In addition, we found that ASXL2 and WTIP are expressed in mouse embryonic epicardial cells, a tissue that is regulated by retinoic acid signaling. Together, these results implicate ASXL2 and WTIP in regulation of retinoic acid signaling during heart development.

Additional sex combs-like family genes are required for normal cardiovascular development.

Congenital heart disease (CHD) is the most common birth defect. However, the majority of CHD cases have unknown etiology. Here we report the identification of ASXL2 and ASXL1, two homologous chromatin factors, as novel regulators of heart development. Asxl2(-/-) fetuses have reduced body weight and display congenital heart malformations including thickened compact myocardium in the left ventricle, membranous ventricular septal defect, and atrioventricular valval stenosis. Although most Asxl2(-/-) animals survive to term, the neonates have patent ductus arteriosus and consequent lung hemorrhage and die soon after birth. Asxl1(-/-) fetuses have reduced body weight and display cleft palate, anophthalmia as well as ventricular septal defects and a failure in lung maturation. From these results, we conclude that normal heart development requires both ASXL proteins. In particular, ASXL2 plays an important role in heart morphogenesis and the transition from fetal to postnatal circulation.

Additional sex combs-like 2 is required for polycomb repressive complex 2 binding at select targets.

Polycomb Group (PcG) proteins are epigenetic repressors of gene expression. The Drosophila Additional sex combs (Asx) gene and its mammalian homologs exhibit PcG function in genetic assays; however, the mechanism by which Asx family proteins mediate gene repression is not well understood. ASXL2, one of three mammalian homologs for Asx, is highly expressed in the mammalian heart and is required for the maintenance of cardiac function. We have previously shown that Asxl2 deficiency results in a reduction in the bulk level of histone H3 lysine 27 trimethylation (H3K27me3), a repressive mark generated by the Polycomb Repressive Complex 2 (PRC2). Here we identify several ASXL2 target genes in the heart and investigate the mechanism by which ASXL2 facilitates their repression. We show that the Asxl2-deficient heart is defective in converting H3K27me2 to H3K27me3 and in removing ubiquitin from mono-ubiquitinated histone H2A. ASXL2 and PRC2 interact in the adult heart and co-localize to target promoters. ASXL2 is required for the binding of PRC2 and for the enrichment of H3K27me3 at target promoters. These results add a new perspective to our understanding of the mechanisms that regulate PcG activity and gene repression.

The chromatin remodeling factor Chd1l is required in the preimplantation embryo.

During preimplantation development, the embryo must establish totipotency and enact the earliest differentiation choices, processes that involve extensive chromatin modification. To identify novel developmental regulators, we screened for genes that are preferentially transcribed in the pluripotent inner cell mass (ICM) of the mouse blastocyst. Genes that encode chromatin remodeling factors were prominently represented in the ICM, including Chd1l, a member of the Snf2 gene family. Chd1l is developmentally regulated and expressed in embryonic stem (ES) cells, but its role in development has not been investigated. Here we show that inhibiting Chd1l protein production by microinjection of antisense morpholinos causes arrest prior to the blastocyst stage. Despite this important function in vivo, Chd1l is non-essential for cultured ES cell survival, pluripotency, or differentiation, suggesting that Chd1l is vital for events in embryos that are distinct from events in ES cells. Our data reveal a novel role for the chromatin remodeling factor Chd1l in the earliest cell divisions of mammalian development.

Maintenance of adult cardiac function requires the chromatin factor Asxl2.

During development and differentiation, cell type-specific chromatin configurations are set up to facilitate cell type-specific gene expression. Defects in the establishment or the maintenance of the correct chromatin configuration have been associated with diseases ranging from leukemia to muscular dystrophy. The heart expresses many chromatin factors, and we are only beginning to understand their roles in heart development and function. We have previously shown that the chromatin regulator Asxl2 is highly expressed in the murine heart both during development and adulthood. In the absence of Asxl2, there is a significant reduction in trimethylation of histone H3 lysine 27 (H3K27), a histone mark associated with lineage-specific silencing of developmental genes. Here we present evidence that Asxl2 is required for the long-term maintenance of ventricular function and for the maintenance of normal cardiac gene expression. Asxl2(-/-) hearts displayed progressive deterioration of ventricular function. By 10 months of age, there was ~37% reduction in fractional shortening in Asxl2(-/-) hearts compared to wild-type. Analysis of the expression of myofibril proteins suggests that Asxl2 is required for the repression of ß-MHC. Asxl2(-/-) hearts did not exhibit hypertrophy, suggesting that the de-repression of ß-MHC was not the result of hypertrophic response. Instead, Asxl2 and the histone methyltansferase Ezh2 co-localize to ß-MHC promoter, suggesting that Asxl2 directly represses ß-MHC. Interrogation of the CardioGenomics database revealed that ASXL2 is down-regulated in the hearts of patients with ischemic or idiopathic dilated cardiomyopathy. We propose that chromatin factors like Asxl2 function in the adult heart to regulate cell type- and stage-specific patterns of gene expression, and the disruption of such regulation may be involved in the etiology and/or development of certain forms of human heart disease.

Epigenetic regulation of cardiac development and function by polycomb group and trithorax group proteins.

Heart disease is a leading cause of death and disability in developed countries. Heart disease includes a broad range of diseases that affect the development and/or function of the cardiovascular system. Some of these diseases, such as congenital heart defects, are present at birth. Others develop over time and may be influenced by both genetic and environmental factors. Many of the known heart diseases are associated with abnormal expression of genes. Understanding the factors and mechanisms that regulate gene expression in the heart is essential for the detection, treatment, and prevention of heart diseases. Polycomb Group (PcG) and Trithorax Group (TrxG) proteins are special families of chromatin factors that regulate developmental gene expression in many tissues and organs. Accumulating evidence suggests that these proteins are important regulators of development and function of the heart as well. A better understanding of their roles and functional mechanisms will translate into new opportunities for combating heart disease.

Mouse genome-wide association and systems genetics identify Asxl2 as a regulator of bone mineral density and osteoclastogenesis.

Significant advances have been made in the discovery of genes affecting bone mineral density (BMD); however, our understanding of its genetic basis remains incomplete. In the current study, genome-wide association (GWA) and co-expression network analysis were used in the recently described Hybrid Mouse Diversity Panel (HMDP) to identify and functionally characterize novel BMD genes. In the HMDP, a GWA of total body, spinal, and femoral BMD revealed four significant associations (-log10P>5.39) affecting at least one BMD trait on chromosomes (Chrs.) 7, 11, 12, and 17. The associations implicated a total of 163 genes with each association harboring between 14 and 112 genes. This list was reduced to 26 functional candidates by identifying those genes that were regulated by local eQTL in bone or harbored potentially functional non-synonymous (NS) SNPs. This analysis revealed that the most significant BMD SNP on Chr. 12 was a NS SNP in the additional sex combs like-2 (Asxl2) gene that was predicted to be functional. The involvement of Asxl2 in the regulation of bone mass was confirmed by the observation that Asxl2 knockout mice had reduced BMD. To begin to unravel the mechanism through which Asxl2 influenced BMD, a gene co-expression network was created using cortical bone gene expression microarray data from the HMDP strains. Asxl2 was identified as a member of a co-expression module enriched for genes involved in the differentiation of myeloid cells. In bone, osteoclasts are bone-resorbing cells of myeloid origin, suggesting that Asxl2 may play a role in osteoclast differentiation. In agreement, the knockdown of Asxl2 in bone marrow macrophages impaired their ability to form osteoclasts. This study identifies a new regulator of BMD and osteoclastogenesis and highlights the power of GWA and systems genetics in the mouse for dissecting complex genetic traits.

Gastrin-releasing peptide signaling alters colon cancer invasiveness via heterochromatin protein 1Hsß.

Epithelial cells lining the adult colon do not normally express gastrin-releasing peptide (GRP) or its receptor (GRPR). In contrast, GRP/GRPR can be aberrantly expressed in colon cancer where they are associated with improved patient survival rates. However, the mechanism of action whereby these proteins mediate their beneficial effects is not known. Heterochromatin protein 1 is an epigenetic modifier of gene transcription for which three different isoforms exist in humans: HP1(Hsα), HP1(Hsß), and HP1(Hsγ). In breast cancer and melanoma, respectively, HP1(Hsα) and HP1(Hsß) have been shown to modulate the aggressiveness of tumor cells in vivo. In contrast, the role of HP1 in colon cancer has not been elucidated, and a mechanism of regulating the expression of any HP1 isoform in any context has not yet been identified. In this article we demonstrate that abrogating GRP/GRPR signaling specifically down-regulates HP1(Hsß) expression and that inhibiting GRPR signaling, or ablating HP1(Hsß) expression, increases colon cancer cell invasiveness in vitro. These findings identify for the first time a signaling pathway regulating heterochromatin protein expression and suggest a mechanism whereby aberrantly expressed GRPR might alter the outcome of patients with colorectal cancer.

Identification of ChIP-seq mapped targets of HP1ß due to bombesin/GRP receptor activation.

Epithelial cells lining the adult colon do not normally express gastrin-releasing peptide (GRP) or its receptor (GRPR). In contrast, GRP/GRPR can be aberrantly expressed in human colorectal cancer (CRC) including Caco-2 cells. We have previously shown that GRPR activation results in the up-regulation of HP1ß, an epigenetic modifier of gene transcription. The aim of this study was to identify the genes whose expression is altered by HP1ß subsequent to GRPR activation. We determined HP1ß binding positions throughout the genome using chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq). After exposure to GRP, we identified 9,625 genomic positions occupied by HP1ß. We performed gene microarray analysis on Caco-2 cells in the absence and presence of a GRPR specific antagonist as well as siRNA to HP1ß. The expression of 97 genes was altered subsequent to GRPR antagonism, while the expression of 473 genes was altered by HP1ß siRNA exposure. When these data were evaluated in concert with our ChIP-seq findings, 9 genes showed evidence of possible altered expression as a function of GRPR signaling via HP1ß. Of these, genomic PCR of immunoprecipitated chromatin demonstrated that GRPR signaling affected the expression of IL1RAPL2, FAM13A, GBE1, PLK3, and SLCO1B3. These findings provide the first evidence by which GRPR aberrantly expressed in CRC might affect tumor progression.

Functional conservation of Asxl2, a murine homolog for the Drosophila enhancer of trithorax and polycomb group gene Asx.

BACKGROUND: Polycomb-group (PcG) and trithorax-group (trxG) proteins regulate histone methylation to establish repressive and active chromatin configurations at target loci, respectively. These chromatin configurations are passed on from mother to daughter cells, thereby causing heritable changes in gene expression. The activities of PcG and trxG proteins are regulated by a special class of proteins known as Enhancers of trithorax and Polycomb (ETP). The Drosophila gene Additional sex combs (Asx) encodes an ETP protein and mutations in Asx enhance both PcG and trxG mutant phenotypes. The mouse and human genomes each contain three Asx homologues, Asx-like 1, 2, and 3. In order to understand the functions of mammalian Asx-like (Asxl) proteins, we generated an Asxl2 mutant mouse from a gene-trap ES cell line. METHODOLOGY/PRINCIPAL FINDINGS: We show that the Asxl2 gene trap is expressed at high levels in specific tissues including the heart, the axial skeleton, the neocortex, the retina, spermatogonia and developing oocytes. The gene trap mutation is partially embryonic lethal and approximately half of homozygous animals die before birth. Homozygotes that survive embryogenesis are significantly smaller than controls and have a shortened life span. Asxl2(-/-) mice display both posterior transformations and anterior transformation in the axial skeleton, suggesting that the loss of Asxl2 disrupts the activities of both PcG and trxG proteins. The PcG-associated histone modification, trimethylation of histone H3 lysine 27, is reduced in Asxl2(-/-) heart. Necropsy and histological analysis show that mutant mice have enlarged hearts and may have impaired heart function. CONCLUSIONS/SIGNIFICANCE: Our results suggest that murine Asxl2 has conserved ETP function and plays dual roles in the promotion of PcG and trxG activity. We have also revealed an unexpected role for Asxl2 in the heart, suggesting that the PcG/trxG system may be involved in the regulation of cardiac function.

Acquisition of essential somatic cell cycle regulatory protein expression and implied activity occurs at the second to third cell division in mouse preimplantation embryos.

It is clear that G1-S phase control is exerted after the mouse embryo implants into the uterus 4.5 days after fertilization (E4.5); null mutants of genes that control cell cycle commitment such as max, rb (retinoblastoma), and dp1 are embryonic lethal after implantation with proliferation phenotypes. But, a number of studies of genes mediating proliferation control in the embryo after fertilization-implantation have yielded confusing results. In order to understand when embryos might first exert G1-S phase regulatory control, we assayed preimplantation mouse embryos for the acquisition of expression of mRNA, protein, and phospho-protein for max, Rb, and DP-1, and for the proliferation-promoting phospho-protein forms of mycC (thr58/ser62) and Rb (ser795). The key findings are that: (1) DP-1 protein was present in the nucleus as early as the four-cell stage onwards, (2) max protein was in the nucleus, suggesting function from the four-cell stage onwards, (3) both mycC and Rb all form protein was present at increasing quantities in the cytoplasm from the 2 cell and 4/8 cell stage, respectively, (4) the phosphorylated form of mycC phospho was present in the nucleus at high levels from the two-cell stage through blastocyst-stage, and (5) the phosphorylated form of Rb was detected at low levels in the two-cell stage embryo and was highly expressed at the 4/8-cell stage through the blastocyst stage. Taken together, these data suggest that activation of mycC phospho/max dimer pairs, (E2F)/DP-1 dimer pairs, and repression of Rb inhibition of cell cycle progression via phosphorylation at ser795 occurs at the earliest stages of embryonic development. In addition, the presence of max, mycC phospho, DP-1, and Rb phospho in the nuclei of embryonic and placental lineage cells in the blastocyst and in trophoblast stem cells suggests that a similar type of cell cycle regulation is present throughout preimplantation development and in both embryonic and extra-embryonic cell lineages.

SAPKgamma/JNK1 and SAPKalpha/JNK2 mRNA transcripts are expressed in early gestation human placenta and mouse eggs, preimplantation embryos, and trophoblast stem cells.

OBJECTIVE: To test early-gestation human placenta, a human trophoblast cell line, mouse eggs, preimplantation embryos, and a mouse trophoblast cell line for the expression of mRNA transcripts for stress-activated protein kinase/c-Jun N-terminal kinase (SAPKgamma/JNK1, SAPKalpha/JNK2, and SAPKbeta/JNK3). DESIGN: Whole RNA was isolated from the tissue sources listed above and control tissues, and reverse transcription-polymerase chain reaction (RT-PCR) was performed to assay for the qualitative and semiquantitative presence of SAPKgamma/JNK1, SAPKalpha/JNK2, and SAPKbeta/JNK3. SETTING: None. PATIENT(S): None. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The presence and magnitude of amplimer amounts in gels or gene hybridization on Affymetrix cDNA arrays of RT-PCR products of reactions for SAPKgamma/JNK1, SAPKalpha/JNK2, and SAPKbeta/JNK3. RESULT(S): SAPKgamma/JNK1 and SAPKalpha/JNK2 mRNA transcripts are present in early-gestation human placenta, a human trophoblast cell line, mouse eggs, preimplantation embryos, and a mouse trophoblast cell line at levels similar to positive control levels. SAPKalpha/JNK2 is expressed at the highest level of the three transcripts in the family. SAPKbeta/JNK3 is present at levels that are 1/100-1/1,000 those of the positive control and in some cases at the apparent level of the negative control (previously measured by the less-sensitive Northern blot analysis). Analysis with an Affymetrix cDNA array suggested that SAPKalpha/JNK2 and 38 kDa mitogen-activated protein kinase had the highest mRNA expression measured for each of three family members. CONCLUSION(S): Mitotic placental trophoblast cell lines and primary conceptus/embryo samples containing early placental trophoblasts express SAPKalpha/JNK2 at higher levels than SAPKgamma/JNK1, but not (only low background levels of) SAPKbeta/JNK3 mRNA transcripts. This suggests that SAPKgamma/JNK1 and SAPKalpha/JNK2 may be important mediators of stress-induced responses in early implanting conceptuses that could mediate embryo loss.

A genome-wide study of gene activity reveals developmental signaling pathways in the preimplantation mouse embryo.

The preimplantation development of the mammalian embryo encompasses a series of critical events: the transition from oocyte to embryo, the first cell divisions, the establishment of cellular contacts, the first lineage differentiation-all the first subtle steps toward a future body plan. Here, we use microarrays to explore gene activity during preimplantation development. We reveal robust and dynamic patterns of stage-specific gene activity that fall into two major phases, one up to the 2-cell stage (oocyte-to-embryo transition) and one after the 4-cell stage (cellular differentiation). The mouse oocyte and early embryo express components of multiple signaling pathways including those downstream of Wnt, BMP, and Notch, indicating that conserved regulators of cell fate and pattern formation are likely to function at the earliest embryonic stages. Overall, these data provide a detailed temporal profile of gene expression that reveals the richness of signaling processes in early mammalian development.

Yeast tRNA as carrier in the isolation of microscale RNA for global amplification and expression profiling.

The characterization of global gene expression patterns of microscale samples is important in many areas of biological and clinical research. The choice of carrier is critical for the efficient isolation and successful amplification of RNA at the nanogram level. Here we show that recovery of nanograms of RNA is significantly higher when carrier linear polyacrylamide is supplemented with carrier tRNA. Reverse transcription and in vitro transcription reactions remain efficient and specific in the presence of carrier tRNA. Finally, comparison of GeneChip array hybridization patterns demonstrates that the presence of carrier tRNA does not cause detectable distortion in global amplification. Taken together, tRNA is a superior carrier for the isolation and global amplification of microscale RNA.