Whole-genome analysis of noncoding genetic variations identifies multiscale regulatory element perturbations associated with Hirschsprung disease.

It is widely recognized that noncoding genetic variants play important roles in many human diseases, but there are multiple challenges that hinder the identification of functional disease-associated noncoding variants. The number of noncoding variants can be many times that of coding variants; many of them are not functional but in linkage disequilibrium with the functional ones; different variants can have epistatic effects; different variants can affect the same genes or pathways in different individuals; and some variants are related to each other not by affecting the same gene but by affecting the binding of the same upstream regulator. To overcome these difficulties, we propose a novel analysis framework that considers convergent impacts of different genetic variants on protein binding, which provides multiscale information about disease-associated perturbations of regulatory elements, genes, and pathways. Applying it to our whole-genome sequencing data of 918 short-segment Hirschsprung disease patients and matched controls, we identify various novel genes not detected by standard single-variant and region-based tests, functionally centering on neural crest migration and development. Our framework also identifies upstream regulators whose binding is influenced by the noncoding variants. Using human neural crest cells, we confirm cell stage-specific regulatory roles of three top novel regulatory elements on our list, respectively in the RET, RASGEF1A, and PIK3C2B loci. In the PIK3C2B regulatory element, we further show that a noncoding variant found only in the patients affects the binding of the gliogenesis regulator NFIA, with a corresponding up-regulation of multiple genes in the same topologically associating domain.

Aberrant DNA Methylation in Acute Myeloid Leukemia and Its Clinical Implications.

Acute myeloid leukemia (AML) is a heterogeneous disease that is characterized by distinct cytogenetic or genetic abnormalities. Recent discoveries in cancer epigenetics demonstrated a critical role of epigenetic dysregulation in AML pathogenesis. Unlike genetic alterations, the reversible nature of epigenetic modifications is therapeutically attractive in cancer therapy. DNA methylation is an epigenetic modification that regulates gene expression and plays a pivotal role in mammalian development including hematopoiesis. DNA methyltransferases (DNMTs) and Ten-eleven-translocation (TET) dioxygenases are responsible for the dynamics of DNA methylation. Genetic alterations of DNMTs or TETs disrupt normal hematopoiesis and subsequently result in hematological malignancies. Emerging evidence reveals that the dysregulation of DNA methylation is a key event for AML initiation and progression. Importantly, aberrant DNA methylation is regarded as a hallmark of AML, which is heralded as a powerful epigenetic marker in early diagnosis, prognostic prediction, and therapeutic decision-making. In this review, we summarize the current knowledge of DNA methylation in normal hematopoiesis and AML pathogenesis. We also discuss the clinical implications of DNA methylation and the current therapeutic strategies of targeting DNA methylation in AML therapy.

Epigenetic dysregulation of leukaemic HOX code in MLL-rearranged leukaemia mouse model.

HOX genes are frequently dysregulated in human leukaemia with the gene rearrangement between mixed lineage leukaemia (MLL) and partner genes. The resultant MLL fusion proteins are known to mediate leukaemia through disruption of the normal epigenetic regulation at the target gene loci. To elucidate the pathogenic role of MLL fusion proteins in HOX dysregulation in leukaemia, we generated a novel haematopoietic lineage-specific Mll-Een knock-in mouse model using a Cre-mediated inversion strategy. The Mll(Een) (/+) invertor mice developed acute myeloid leukaemia, with organomegaly of the spleen, liver and mesenteric lymph nodes caused by infiltration of blast cells. Using Mll-Een-expressing leukaemic cell lines derived from bone marrow of Mll(Een) (/+) mutant mice, we showed that induction of Hox genes in leukaemic cells was associated with hypomethylated promoter regions and an aberrant active chromatin state at the Hox loci. Knock-down of Prmt1 was insufficient to reverse the active chromatin status and the hypomethylated Hox loci, suggesting that Prmt1-mediated histone arginine methylation was only partially involved in the maintenance of Hox expression in leukaemic cells. Furthermore, in vivo analysis of bone marrow cells of Mll(Een) (/+) mice revealed a Hox expression profile similar to that of wild-type haematopoietic stem cells. The leukaemic Hox profile was highly correlated with aberrant hypomethylation of Hox promoters in the mutant mice, which highlights the importance of DNA methylation in leukaemogenic mechanisms induced by MLL fusion proteins. Our results point to the involvement of dynamic epigenetic regulations in the maintenance of the stem cell-like HOX code that initiates leukaemic stem cells in MLL-rearranged leukaemia. This provides insights for the development of alternative strategies for leukaemia treatment.

Cytokine combinations on the potential for ex vivo expansion of murine hematopoietic stem cells.

Hematopoietic stem cell (HSC) is a rare cell population, which is capable of self-renewal and differentiation to all blood lineages. The clinical potential of HSCs for treating hematological disorders has led to the use of cytokine stimulation for ex vivo expansion. However, little is known about the molecular features of the HSC populations expanded under different cytokine combinations. We studied the expansion of murine HSCs cultured with six different cytokine combinations under serum-containing or serum-free conditions for 14days. We found that all the cytokine combinations promoted expansion of murine HSCs. Although SCF/IL-3/IL-6 induced the highest expansion of the immunophenotypic Lineage(-)Sca-1(+)c-Kit(+) (LSK) cells at day 14, over 90% of them were FcεRIα(+) mast cells. In contrast, the serum-free medium with SCF/Flt3-L/IL-11 effectively promoted the expansion of LSK/FcεRIα(-) HSCs by over 50-fold. HSCs expanded by SCF/Flt3-L/IL-11 combination formed compact hematopoietic colonies and demonstrated a higher degree of multipotency compared to the HSCs cultured with other cytokine combinations. Surprisingly, despite the same LSK/FcεRIα(-) immunophenotype, HSCs cultured with different cytokine combinations demonstrated differential patterns of hematopoietic gene expression. HSCs cultured with SCF/Flt3-L/IL-11 maintained a transcription profile resembling that of freshly isolated HSCs. We propose that serum-free medium supplemented with SCF/Flt3-L/IL-11 is the optimal culture condition to maintain the stemness of ex vivo expanded HSCs. This study used molecular characterization of cytokine-expanded murine HSCs to facilitate the selection of cytokine combinations that could induce fully competent HSC for clinical applications.

Global mapping of DNA methylation in mouse promoters reveals epigenetic reprogramming of pluripotency genes.

DNA methylation patterns are reprogrammed in primordial germ cells and in preimplantation embryos by demethylation and subsequent de novo methylation. It has been suggested that epigenetic reprogramming may be necessary for the embryonic genome to return to a pluripotent state. We have carried out a genome-wide promoter analysis of DNA methylation in mouse embryonic stem (ES) cells, embryonic germ (EG) cells, sperm, trophoblast stem (TS) cells, and primary embryonic fibroblasts (pMEFs). Global clustering analysis shows that methylation patterns of ES cells, EG cells, and sperm are surprisingly similar, suggesting that while the sperm is a highly specialized cell type, its promoter epigenome is already largely reprogrammed and resembles a pluripotent state. Comparisons between pluripotent tissues and pMEFs reveal that a number of pluripotency related genes, including Nanog, Lefty1 and Tdgf1, as well as the nucleosome remodeller Smarcd1, are hypomethylated in stem cells and hypermethylated in differentiated cells. Differences in promoter methylation are associated with significant differences in transcription levels in more than 60% of genes analysed. Our comparative approach to promoter methylation thus identifies gene candidates for the regulation of pluripotency and epigenetic reprogramming. While the sperm genome is, overall, similarly methylated to that of ES and EG cells, there are some key exceptions, including Nanog and Lefty1, that are highly methylated in sperm. Nanog promoter methylation is erased by active and passive demethylation after fertilisation before expression commences in the morula. In ES cells the normally active Nanog promoter is silenced when targeted by de novo methylation. Our study suggests that reprogramming of promoter methylation is one of the key determinants of the epigenetic regulation of pluripotency genes. Epigenetic reprogramming in the germline prior to fertilisation and the reprogramming of key pluripotency genes in the early embryo is thus crucial for transmission of pluripotency.

Histone demethylase JMJD2B/KDM4B regulates transcriptional program via distinctive epigenetic targets and protein interactors for the maintenance of trophoblast stem cells.

Trophoblast stem cell (TSC) is crucial to the formation of placenta in mammals. Histone demethylase JMJD2 (also known as KDM4) family proteins have been previously shown to support self-renewal and differentiation of stem cells. However, their roles in the context of the trophoblast lineage remain unclear. Here, we find that knockdown of Jmjd2b resulted in differentiation of TSCs, suggesting an indispensable role of JMJD2B/KDM4B in maintaining the stemness. Through the integration of transcriptome and ChIP-seq profiling data, we show that JMJD2B is associated with a loss of H3K36me3 in a subset of embryonic lineage genes which are marked by H3K9me3 for stable repression. By characterizing the JMJD2B binding motifs and other transcription factor binding datasets, we discover that JMJD2B forms a protein complex with AP-2 family transcription factor TFAP2C and histone demethylase LSD1. The JMJD2B-TFAP2C-LSD1 complex predominantly occupies active gene promoters, whereas the TFAP2C-LSD1 complex is located at putative enhancers, suggesting that these proteins mediate enhancer-promoter interaction for gene regulation. We conclude that JMJD2B is vital to the TSC transcriptional program and safeguards the trophoblast cell fate via distinctive protein interactors and epigenetic targets.

Structural mechanism of bivalent histone H3K4me3K9me3 recognition by the Spindlin1/C11orf84 complex in rRNA transcription activation.

Spindlin1 is a unique multivalent epigenetic reader that facilitates ribosomal RNA transcription. In this study, we provide molecular and structural basis by which Spindlin1 acts in complex with C11orf84 to preferentially recognize non-canonical bivalent mark of trimethylated lysine 4 and lysine 9 present on the same histone H3 tail (H3K4me3K9me3). We demonstrate that C11orf84 binding stabilizes Spindlin1 and enhances its association with bivalent H3K4me3K9me3 mark. The functional analysis suggests that Spindlin1/C11orf84 complex can displace HP1 proteins from H3K4me3K9me3-enriched rDNA loci, thereby facilitating the conversion of these poised rDNA repeats from the repressed state to the active conformation, and the consequent recruitment of RNA Polymerase I for rRNA transcription. Our study uncovers a previously unappreciated mechanism of bivalent H3K4me3K9me3 recognition by Spindlin1/C11orf84 complex required for activation of rRNA transcription.

Murine Mesenchymal Stromal Cells Retain Biased Differentiation Plasticity Towards Their Tissue of Origin.

Mesenchymal stromal/stem cells (MSCs) reside in many human tissues and comprise a heterogeneous population of cells with self-renewal and multi-lineage differentiation potential, making them useful in regenerative medicine. It remains inconclusive whether MSCs isolated from different tissue sources exhibit variations in biological features. In this study, we derived MSCs from adipose tissue (AT-MSC) and compact bone (CB-MSC). We found that early passage of MSCs was readily expandable ex vivo, whereas the prolonged culture of MSCs showed alteration of cell morphology to fibroblastoid and reduced proliferation. CB-MSCs and AT-MSCs at passage 3 were CD29+, CD44+, CD105+, CD106+, and Sca-1+; however, passage 7 MSCs showed a reduction of MSC markers, indicating loss of stem cell population after prolonged culturing. Strikingly, CB-MSC was found more efficient at undergoing osteogenic differentiation, while AT-MSC was more efficient to differentiate into adipocytes. The biased differentiation pattern of MSCs from adipogenic or osteogenic tissue source was accompanied by preferential expression of the corresponding lineage marker genes. Interestingly, CB-MSCs treated with DNA demethylation agent 5-azacytidine showed enhanced osteogenic and adipogenic differentiation, whereas the treated AT-MSCs are less competent to differentiate. Our results suggest that the epigenetic state of MSCs is associated with the biased differentiation plasticity towards its tissue of origin, proposing a mechanism related to the retention of epigenetic memory. These findings facilitate the selection of optimal tissue sources of MSCs and the ex vivo expansion period for therapeutic applications.

Histone lysine demethylase KDM5B maintains chronic myeloid leukemia via multiple epigenetic actions.

The histone lysine demethylase KDM5 family is implicated in normal development and stem cell maintenance by epigenetic modulation of histone methylation status. Deregulation of the KDM5 family has been reported in various types of cancers, including hematological malignancies. However, their transcriptional regulatory roles in the context of leukemia remain unclear. Here, we find that KDM5B is strongly expressed in normal CD34+ hematopoietic stem/progenitor cells and chronic myeloid leukemia (CML) cells. Knockdown of KDM5B in K562 CML cells reduced leukemia colony-forming potential. Transcriptome profiling of KDM5B knockdown K562 cells revealed the deregulation of genes involved in myeloid differentiation and Toll-like receptor signaling. Through the integration of transcriptome and ChIP-seq profiling data, we show that KDM5B is enriched at the binding sites of the GATA and AP-1 transcription factor families, suggesting their collaborations in the regulation of transcription. Even though the binding of KDM5B substantially overlapped with H3K4me1 or H3K4me3 mark at gene promoters, only a small subset of the KDM5B targets showed differential expression in association with the histone demethylation activity. By characterizing the interacting proteins in K562 cells, we discovered that KDM5B recruits protein complexes involved in the mRNA processing machinery, implying an alternative epigenetic action mediated by KDM5B in gene regulation. Our study highlights the oncogenic functions of KDM5B in CML cells and suggests that KDM5B is vital to the transcriptional regulation via multiple epigenetic mechanisms.

Reactive oxygen species activate differentiation gene transcription of acute myeloid leukemia cells via the JNK/c-JUN signaling pathway.

Reactive oxygen species (ROS) and altered cellular redox status are associated with many malignancies. Acute myeloid leukemia (AML) cells are maintained at immature state by differentiation blockade, which involves deregulation of transcription factors in myeloid differentiation. AML cells can be induced to differentiate by phorbol-12-myristate-13-acetate (PMA), which possesses pro-oxidative activity. However, the signaling events mediated by ROS in the activation of transcriptional program during AML differentiation has not been fully elucidated. Here, we investigated AML cell differentiation by treatment with PMA and ROS scavenger N-acetyl-l-cysteine (NAC). We observed elevation of intracellular ROS level in the PMA-treated AML cells, which correlated with differentiated cell morphology and increased CD11b+ mature cell population. The effect of PMA can be abolished by NAC co-treatment, supporting the involvement of ROS in the process. Moreover, we demonstrated that short ROS elevation mediated cell cycle arrest, but failed to activate myeloid gene transcription; whereas prolonged ROS elevation activated JNK/c-JUN signaling pathway. Inhibition of JNK suppressed the expression of key myeloid transcriptional regulators c-JUN, SPI-1 and MAFB, and prevented AML cells from undergoing terminal differentiation. These findings provide new insights into the crucial role of JNK/c-Jun signaling pathway in the activation of transcriptional program during ROS-mediated AML differentiation.

Histone deacetylase inhibitors induce leukemia gene expression in cord blood hematopoietic stem cells expanded ex vivo.

Umbilical cord blood is a valuable source of hematopoietic stem cells. While cytokine stimulation can induce ex vivo hematopoietic cell proliferation, attempts have been made to use epigenetic-modifying agents to facilitate stem cell expansion through the modulation of cellular epigenetic status. However, the potential global effect of these modifying agents on epigenome raises concerns about the functional normality of the expanded cells. We studied the ex vivo expansion of cord blood hematopoietic stem and progenitor cells (HSPCs) by histone deacetylase (HDAC) inhibitors, trichostatin A and valproic acid. Treatment with HDAC inhibitors resulted in mild expansion of the total hematopoietic cell number when compared with cytokine stimulated sample. Nevertheless, we observed 20-30-fold expansion of the CD34+ CD38- HSPC population. Strikingly, cord blood cells cultured with HDAC inhibitors exhibited aberrant expression of leukemia-associated genes, including CDKN1C, CEBPα, HOXA9, MN1, and DLK1. Our results thus suggest that the expansion of HSPCs by this approach may provoke a pre-leukemic cell state. We propose that the alteration of epigenome by HDAC inhibitors readily expands cord blood HSPC population through the re-activation of the leukemia gene transcription. The present study provides an assessment of the leukemogenic potential of HSCs expanded ex vivo using HDAC inhibitors for clinical applications.

Epigenetic regulation of pluripotent genes mediates stem cell features in human hepatocellular carcinoma and cancer cell lines.

Activation of the stem cell transcriptional circuitry is an important event in cancer development. Although cancer cells demonstrate a stem cell-like gene expression signature, the epigenetic regulation of pluripotency-associated genes in cancers remains poorly understood. In this study, we characterized the epigenetic regulation of the pluripotency-associated genes NANOG, OCT4, c-MYC, KLF4, and SOX2 in a variety of cancer cell lines and in primary tumor samples, and investigated the re-activation of pluripotency regulatory circuits in cancer progression. Differential patterns of DNA methylation, histone modifications, and gene expression of pluripotent genes were demonstrated in different types of cancers, which may reflect their tissue origins. NANOG promoter hypomethylation and gene upregulation were found in metastatic human liver cancer cells and human hepatocellular carcinoma (HCC) primary tumor tissues. The upregulation of NANOG, together with p53 depletion, was significantly associated with clinical late stage of HCC. A pro-metastatic role of NANOG in colon cancer cells was also demonstrated, using a NANOG-overexpressing orthotopic tumor implantation mouse model. Demethylation of NANOG promoter was observed in CD133+(high) cancer cells. In accordance, overexpression of NANOG resulted in an increase in the population of CD133+(high) cells. In addition, we demonstrated a cross-regulation between OCT4 and NANOG in cancer cells via reprogramming of promoter methylation. Taken together, epigenetic reprogramming of NANOG can lead to the acquisition of stem cell-like properties. These results underscore the restoration of pluripotency circuits in cancer cells as a potential mechanism for cancer progression.

Activation of Ras-dependent Elk-1 activity by MLL-AF4 family fusion oncoproteins.

OBJECTIVE: Mixed lineage leukemia (MLL) gene rearrangement is commonly observed in human leukemias. Many of the resultant MLL fusion proteins are found correlated with Ras signaling. Nevertheless, Ras mutations have only been reported in a small subset of MLL-rearranged leukemia. With the potential of developing new therapeutic regimens targeting Ras signaling pathway, we studied the role of MLL-AF4 family fusions and MLL-septin family fusions in the activation of Ras signaling in leukemogenesis. MATERIALS AND METHODS: Elk-1-driven luciferase reporter system was used to study the role of MLL-AF4, MLL-AF5q31, MLL-LAF4, MLL-CDCrel, MLL-MSF, and MLL-Septin 6 in the activation of Ras signaling. Dominant negative Ras S17N mutant and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) inhibitor U0126 were employed to demonstrate the involvement of Ras and MEK in this transactivation event. The activation of endogenous Ras/MEK signaling pathway by MLL fusion proteins in leukemia cell lines was also addressed by immunoblot analysis and small interfering RNA knockdown approach. RESULTS: We demonstrated that MLL-AF4, MLL-AF5q31, and MLL-LAF4 activated Elk-1 transcription factor, one of the major downstream effectors of Ras. This activation was abolished in the presence of dominant negative Ras or MEK inhibitor U0126, indicating the requirements of Ras and MEK. We further showed that endogenous MEK is phosphorylated in a MLL-AF4-expressing leukemia cell line, whereas depletion of MLL-AF4 by small interfering RNA reduced the phospho-MEK level. CONCLUSION: Our findings suggest that MLL-AF4 family fusion oncoproteins can activate Elk-1 through Ras/MEK/extracellular signal-regulated kinase (ERK) pathway and strongly support the role of Ras signaling in the pathogenesis of MLL-rearranged leukemia.

Epigenetic memory of an active gene state depends on histone H3.3 incorporation into chromatin in the absence of transcription.

The remarkable stability of gene expression in somatic cells is exemplified by the way memory of an active gene state is retained when an endoderm cell nucleus is transplanted to an enucleated egg. Here we analyse the mechanism of a similar example of epigenetic memory. We find that memory can persist through 24 cell divisions in the absence of transcription and applies to the expression of the myogenic gene MyoD in non-muscle cell lineages of nuclear transplant embryos. We show that memory is not explained by the methylation of promoter DNA. However, we demonstrate that epigenetic memory correlates with the association of histone H3.3 with the MyoD promoter in embryos that display memory but not in those where memory has been lost. The association of a mutated histone H3.3 (H3.3 E4, which lacks the methylatable H3.3 lysine 4) with promoter DNA eliminates memory, indicating a requirement of H3.3 K4 for memory. We also show that overexpression of H3.3 can enhance memory in transplanted nuclei. We therefore conclude that the association of histone H3.3 with the MyoD promoter makes a necessary contribution to this example of memory. Hence, we suggest that epigenetic memory helps to stabilize gene expression in normal development; it might also help to account for the inefficient reprogramming in some transplanted nuclei.

Epigenetic restriction of embryonic cell lineage fate by methylation of Elf5.

Mouse ES cells can differentiate into all three germ layers of the embryo but are generally excluded from the trophoblast lineage. Here we show that ES cells deficient in DNA methylation can differentiate efficiently into trophoblast derivatives. In a genome-wide screen we identified the transcription factor Elf5 as methylated and repressed in ES cells, and hypomethylated and expressed in TS and methylation-deficient ES cells. Elf5 creates a positive-feedback loop with the TS cell determinants Cdx2 and Eomes that is restricted to the trophoblast lineage by epigenetic regulation of Elf5. Importantly, the late-acting function of Elf5 allows initial plasticity and regulation in the early blastocyst. Thus, Elf5 functions as a gatekeeper, downstream of initial lineage determination, to reinforce commitment to the trophoblast lineage or to abort this pathway in epiblast cells. This epigenetic restriction of cell lineage fate provides a molecular mechanism for Waddington's concept of canalization of developmental pathways.

cDNA microarray analysis of early gene expression profiles associated with hepatitis B virus X protein-mediated hepatocarcinogenesis.

Chronic hepatitis B virus (HBV) infection is one of the major causes of hepatocellular carcinoma. HBV encodes an oncogenic hepatitis B virus X protein (HBx), which can transactivate host cell transcriptional machinery and mediate cellular transformation. To disclose the early genetic response in HBx-mediated transformation process, we constructed a conditional HBx-expressing hepatocyte cell line, which allows us to compare the gene expression profiles under controllable HBx induction. A cDNA microarray containing more than 8700 mouse genes and ESTs was utilized to examine the gene expression profiles. We identified 260 candidate genes and 259 ESTs which have shown aberrant expression under HBx induction. Most of them are involved in signal transduction pathway, cell cycle control, metastasis, transcriptional regulation, immune response, and metabolism. These results provide additional insight into early cellular targets of HBx, which could give us a better understanding of the function of HBx and their progressive changes during HBx-mediated hepatocarcinogenesis.