Regulation Of Hox Gene Expression Research Articles

Nuclear Condensates Are a Therapeutic Vulnerability in NPM1-Mutant Acute Myeloid Leukemia

The development of targeted therapies against drivers of malignancy requires a robust mechanistic understanding of the pathogenic axis underlying disease. Several subtypes of acute myeloid leukemias (AML), such as those driven by mutations in IDH1/2, FLT3, and KMT2A, have benefitted from the development of novel therapeutics that disrupt their transcriptional program. In contrast, there are no approved therapies that target the mutant version of Nucleophosmin (NPM1), which is the most common driver in adult AML. Mutations in NPM1 result in export of the mutant protein (NPM1c) to the cytoplasm, prompting the suggestion that aberrant localization leads to neomorphic and pathogenic functions of NPM1c in the cytoplasm. In parallel, recent data from several groups has suggested that NPM1c directly regulates gene expression within the nucleus. Despite this progress, a well-defined mechanism for NPM1c-mediated gene regulation is lacking, hindering the development of NPM1c-targeted therapies. To investigate the function of NPM1c, we generated over a dozen AML cell lines expressing unique NPM1c truncations and characterized their impact on the leukemic transcriptome and cell morphology. To our surprise, we found that although the majority of these truncated proteins reside in the cytoplasm, most were unable to recapitulate the transcriptional program driven by full-length NPM1c, and cells subsequently underwent differentiation and growth arrest. We further explored the chromatin binding ability of these truncated proteins to key genomic regions such as the HOXA cluster and MEIS1 through CUT&RUN analysis. Next, we identified a region of the NPM1c protein critical for active nuclear import (NLS). Strikingly, disruption of this region enforces cytoplasmic sequestration of the truncated NPM1c protein. However, cells harboring this truncation fail to maintain leukemic gene expression and differentiate. Together these results suggest that aberrant localization and/or shuttling of NPM1c between cellular compartments is insufficient to maintain leukemic gene expression and cell-stemness. We next considered whether NPM1c had a de-novo function that may explain its role in gene regulation. We have recently shown that NPM1c forms phase-separated condensates in the nucleus that regulate HOX gene expression and are necessary for stem-ness. These NPM1c condensates recruit multiple gene regulatory factors including XPO1, NUP98, and KMT2A (Figure 1). Through selective degradation of NPM1c, or pharmacological inhibition of XPO1, we found that disruption of nuclear condensates abrogates recruitment of these protein co-factors. We further explore the effect of drug compounds known to inhibit NPM1-mutant AML on condensate behavior and chromatin binding. This condensate behavior is a defining feature of heterotypic phase separation, a process in which two or more biomolecules form multivalent interactions to create a condensate. Importantly, we found that significant overexpression of NPM1c causes an imbalance in the ratio between NPM1c and its binding partners, disrupting normal condensate formation, and causing subsequent differentiation and growth arrest. Collectively, these results suggest that therapeutic targeting of NPM1c, or a host of interacting partners, may disrupt condensate formation and the leukemic transcriptional program, providing an exciting new therapeutic vulnerability in NPM1-mutant AML.

Maternal SMCHD1 regulates Hox gene expression and patterning in the mouse embryo

Parents transmit genetic and epigenetic information to their offspring. Maternal effect genes regulate the offspring epigenome to ensure normal development. Here we report that the epigenetic regulator SMCHD1 has a maternal effect on Hox gene expression and skeletal patterning. Maternal SMCHD1, present in the oocyte and preimplantation embryo, prevents precocious activation of Hox genes post-implantation. Without maternal SMCHD1, highly penetrant posterior homeotic transformations occur in the embryo. Hox genes are decorated with Polycomb marks H2AK119ub and H3K27me3 from the oocyte throughout early embryonic development; however, loss of maternal SMCHD1 does not deplete these marks. Therefore, we propose maternal SMCHD1 acts downstream of Polycomb marks to establish a chromatin state necessary for persistent epigenetic silencing and appropriate Hox gene expression later in the developing embryo. This is a striking role for maternal SMCHD1 in long-lived epigenetic effects impacting offspring phenotype.

Nuclear-localized human respiratory syncytial virus NS1 protein modulates host gene transcription.

SUMMARYHuman respiratory syncytial virus (RSV) is a common cause of lower respiratory tract infections in the pediatric, elderly, and immunocompromised individuals. RSV non-structural protein NS1 is a known cytosolic immune antagonist, but how NS1 modulates host responses remains poorly defined. Here, we observe NS1 partitioning into the nucleus of RSV-infected cells, including the human airway epithelium. Nuclear NS1 coimmunoprecipitates with Mediator complex and is chromatin associated. Chromatin-immunoprecipitation demonstrates enrichment of NS1 that overlaps Mediator and transcription factor binding within the promoters and enhancers of differentially expressed genes during RSV infection. Mutation of the NS1 C-terminal helix reduces NS1 impact on host gene expression. These data suggest that nuclear NS1 alters host responses to RSV infection by binding at regulatory elements of immune response genes and modulating host gene transcription. Our study identifies another layer of regulation by virally encoded proteins that shapes host response and impacts immunity to RSV.

Abstract 3089: Study of HOX genes in the stem cell origin of CRC

Abstract Our goal is to ascertain how HOX-regulatory mechanisms play a role in the SC origin of colorectal cancer (CRC). While HOX genes encode master regulatory transcription factors that regulate stem cells (SCs), their role in cancer etiology is under-studied. We and others find that HOX genes are aberrantly expressed in CRC. We previously reported that HOXA4 and HOXD10 along with aldehyde dehydrogenase (ALDH) and the other components of the retinoic acid (RA) signaling pathway are selectively expressed in human colon SCs. That RA signaling regulates HOX expression indicates that RA regulation of HOX gene expression mainly occurs through ALDH+ SCs in the colon. We hypothesize that specific HOX genes regulate growth and differentiation of SCs in human colon and HOX gene dysregulation leads to decreased differentiation and SC overpopulation. Indeed, our bioinformatics analysis shows that many HOX genes are overexpressed in CRCs, which parallels the overpopulation of ALDH+ SC that occurs during CRC tumorigenesis. We are using immunohistochemical mapping to measure proportions of SCs and HOX-positive cells in normal and malignant human colon tissues. We find that specific HOX proteins have increased expression levels in malignant colon tissues. In addition, immunofluorescence co-staining of human colon tissues is used to validate HOX gene expression in ALDH+ SCs. We are also evaluating the effects of all-trans-RA (ATRA) on HOX gene expression and on cellular proliferation and differentiation using CRC cell lines. Our results show that ATRA treatment of HT29 CRC cells leads to reduced proliferation and decreased expression of select HOX genes. These results indicate that HOX genes like HOXA4, HOXA9, and HOXD10, are expressed colonic SCs, and are regulated by RA signaling. Overall, the overexpression of HOX genes in cancer SCs appears to contribute to the SC origin of CRC. Citation Format: Brian Osmond, Caroline O. Facey, Lynn M. Opdenaker, Bruce M. Boman. Study of HOX genes in the stem cell origin of CRC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3089.

Pontin Functions as A Transcriptional Co-activator for Retinoic Acid-induced HOX Gene Expression

Pontin is a AAA+ ATPase protein that has functions in various biological contexts including gene transcription regulation, chromatin remodeling, DNA damage sensing and repair, as well as assembly of protein and ribonucleoprotein complexes. Pontin is known to regulate the transcription of several important signaling pathways, including Wnt signaling. However, its role in early embryonic signaling regulation remains unclear. Retinoic acid (RA) signaling plays a central role in vertebrate development. Using an in vivo biotin tagging technology, we mapped the genome-wide binding pattern of Pontin before and after RA-induced differentiation in the pluripotent embryo carcinoma cell line NTERA-2. Biotin ChIP-seq revealed significant changes in genome-wide Pontin binding sites upon RA stimulation. We also identified a substantial amount of overlapping binding peaks between Pontin and RARα, especially on all of the HOX gene loci (A-D clusters). Pontin knockdown experiments showed that its chromatin binding at the HOX gene clusters is required for RA-induced HOX gene expression. Furthermore, we performed Global Run-On sequencing (GRO-seq) to map de novo transcripts genome-wide and found that Pontin knockdown significantly diminished nascent HOX gene transcripts, indicating that Pontin regulates HOX gene expression at the transcriptional level. Finally, proteomic analysis demonstrated that Pontin associates with chromatin organization/remodeling complexes and various other functional complexes. Altogether, we have demonstrated that Pontin is a critical transcriptional co-activator for RA-induced HOX gene activation.

ERK‐mediated Phosphorylation of the Egr Transcriptional Coregulator NAB2 at Multiple Sites

The early growth response (Egr) family of transcription factors comprises five members, Egr1‐4 and Wilms Tumor 1 (WT1), which play roles in various cell behaviors including proliferation, apoptosis, and differentiation in a cell type‐ and stimulus‐specific manner. Roles for Egr1‐3 are particularly well‐documented in the nervous system. Egr1 and 3 contribute to learning and memory through regulation of genes that mediate synaptic plasticity and long‐term potentiation. Egr2 controls hindbrain development through regulation of Hox gene expression and drives peripheral nerve myelination through, at least in part, activation of the gene encoding myelin protein zero in Schwann cells. Egr1 and 2 also contribute to transcriptional network that drives neuronal differentiation of PC12 cells. However, the mechanisms that regulate Egr1‐3 activities in the nervous system are not fully understood. One mechanism of regulation involves protein‐protein interactions with co‐regulators NAB1 and 2 through a conserved R1 domain in Egr1‐3. Most reports demonstrate that NABs repress Egr1‐3 transactivation activity when bound, however, NABs can also enhance transactivation in a target gene‐specific manner. The mechanisms controlling the effect of NABs on Egr activities are not clear. Here, we provide evidence that NAB2 regulation may involve its phosphorylation at several sites by ERK. Using PC12 cells as a model system, our data demonstrate that nerve growth factor (NGF) induces a NAB2 mobility shift in SDS‐PAGE within 10 minutes of treatment. The mobility shift was reversed when lysates from NGF‐treated cells were treated with lambda phosphatase, suggesting the shift is due to phosphorylation. Inhibition of ERK signaling during NGF treatment blocked the mobility shift, while recombinant ERK phosphorylated recombinant NAB2 in an in vitro kinase assay, together indicating that direct phosphorylation of NAB2 by ERK may be responsible for the NAB2 mobility shift in response to NGF. Nine potential ERK phosphorylation sites were identified in NAB2, which were mutated individually to alanine within a NAB2 expression construct to evaluate phosphorylation at those sites based on their mobility in SDS‐PAGE. Of the nine mutants, three exhibited clear changes in NAB2 mobility, which were additive in double and triple mutants for those sites. Ongoing experiments are evaluating the effect of NAB2 phosphorylation on its localization and regulation of Egr1‐3.Support or Funding InformationBridgewater State University Undergraduate Research Program

Why a Constant Number of Vertebrae? Digital Control of Segmental Identity during Vertebrate Development: The Somite Cycle Controls a Digital, Chromatin-Based Counter That Defines Segmental Identity and Body Plans in Vertebrate Animals.

It is not understood how the numbers and identities of vertebrae are controlled during mammalian development. The remarkable robustness and conservation of segmental numbers may suggest the digital nature of the underlying process. The study proposes a mechanism that allows cells to obtain and store the segmental information in digital form, and to produce a pattern of chromatin accessibility that in turn regulates Hox gene expression specific to the metameric segment. The model requires that a regulatory element be present such that the number of occurrences of the motif between two consecutive Hox genes equals the number of segments under the control of the anterior gene. This is true for the recently discovered hydroxyl radical cleavage 3bp-periodic (HRC3) motif, associated with histone modifications and developmental genes. The finding not only allows the correct prediction of the numbers of segments using only sequence information, but also resolves the 40-year-old enigma of the function of temporal and spatial collinearity of Hox genes. The logic of the mechanism is illustrated in the attached animated video. How different aspects of the proposed mechanism can be tested experimentally is also discussed.

Histone Demethylase Activity of Utx Is Essential for Viability and Regulation of HOX Gene Expression in Drosophila.

The trimethylation of histone H3 at lysine 27 (H3K27me3) by Polycomb Repressive Complex 2 (PRC2) is essential for the repression of Polycomb target genes. However, the role of enzymatic demethylation of H3K27me3 by the KDM6-family demethylases Utx, Uty, and JmjD3 is less clear. Studies in both mice and worms led to the proposal that KDM6 proteins, but not their H3K27me3 demethylase activity, is critical for normal development. Here, we investigated the requirement of the demethylase activity of the single KDM6 family member Utx in Drosophila. We generated Drosophila expressing a full-length but catalytically inactive Utx protein and found that these mutants show the same phenotypes as animals lacking the Utx protein. Specifically, animals lacking maternally deposited active Utx demethylase in the early embryo show stochastic loss of HOX gene expression that appears to be propagated in a clonal fashion. This suggests that Utx demethylase activity is critical for the removal of ectopic H3K27 trimethylation from active HOX genes during the onset of zygotic gene transcription, and thereby prevents the inappropriate installment of long-term repression by Polycomb. Conversely, maternally deposited catalytically active Utx protein suffices to permit animals that lack zygotic expression of enzymatically active Utx to develop into morphologically normal adults, which eclose from the pupal case but die shortly thereafter. Utx demethylase activity is therefore also essential to sustain viability in adult flies. Together, these analyses identify the earliest embryonic stages and the adult stage as two phases during the Drosophila life cycle that critically require H3K27me3 demethylase activity.

Abstract 3042: MIR142 loss-of-function mutations promote leukemogenesis through derepression of ASH1L resulting in increased HOX gene expression

Abstract MIR142 mutations have been identified in acute myeloid leukemia (AML) and non-Hodgkins lymphoma. In AML, all MIR142 mutations localize to the miR-142-3p seed sequence. We show that mutated MIR142 is unable to suppress several well-known targets of miR-142-3p. Interestingly, the mutations of miR-142-3p result in their preferential loading into the RNA-induced silencing complex, leading to the degradation of miR-142-5p. Accordingly, miR-142-5p expression is decreased in MIR142 mutated AML. Hence, MIR142 mutations in AML disrupt both miR-142-3p/5p functions. Thus, we modeled the effect of MIR142 mutations on hematopoiesis using Mir142-/- mice. We show that loss of miR142 results in significant increases in myeloid hematopoietic stem/progenitor cells (HSPCs), including granulocyte-macrophage progenitors, myeloid-biased multipotent progenitors (CD150- CD48+ Flk2- Kit+ Sca+ lineage-) and CD229- myeloid-biased HSCs (CD150+ CD48- Kit+ Sca+ lineage-). In contrast, there are significant decreases of megakaryocyte-erythroid progenitors and erythroid precursors. Although the number of HSCs is normal in Mir142-/- mice, HSC transplantation suggest that they are myeloid-biased. In AML, MIR142 mutations are commonly found in conjunction with mutations of IDH1/2. To assess the importance of this association, we transduced wildtype or Mir142-/- HSPCs with retrovirus expressing IDH2 R172K and then transplanted into lethally irradiated recipients. Expression of IDH2 R172K alone was sufficient to induce a lethal myeloproliferative neoplasm (MPN). In contrast, Mir142-/- alone did not result in MPN. However, loss of Mir142 cooperates with IDH2 R172K to produce a more severe MPN, with increased CD34+ blasts and more severe anemia. Moreover, secondary transplantation shows that Mir142-/- x IDH2 R172K cells but not IDH2 R172K cells efficiently engraft and induce MPN, suggesting that loss of miR142 increases leukemia-initiating activity. We identify the histone methyltransferase ASH1L as a target gene of miR142 that contributes to altered hematopoiesis in Mir142-/- mice. The 3’-untranslated region of ASH1L has four miR-142-3p binding sites, and luciferase reporter assay shows that miR142 suppresses its translation by 80%. Consistent with this observation, Ashl1 protein expression is 3-fold higher in Mir142-/- bone marrow. ASH1L is a key positive regulator of HOX gene expression. Accordingly, we observed markedly (5-10 fold) increased HoxA9/A10 expression in myeloid progenitors in Mir142-/- mice. Likewise, HoxA9/A10 expression is increased in CD34+ blasts from Mir142-/- x IDH2 R172K transplanted mice. Of note, increased HoxA9 expression has been shown to cooperate with mutant IDH1 to induce AML in mice. Together, these findings support a model in which loss-of-function mutations of MIR142 contribute to hematopoietic malignancies by derepressing ASH1L and inducing HOXA9/10 gene expression. Citation Format: Juo-Chin Yao, Terrence N. Wong, Maria Trissal, Rahul Ramaswamy, Yaping Sun, Pavan R. Reddy, Daniel C. Link. MIR142 loss-of-function mutations promote leukemogenesis through derepression of ASH1L resulting in increased HOX gene expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3042. doi:10.1158/1538-7445.AM2017-3042

Regulation of Hox gene expression during anteroposterior patterning involves collinear Hox-Hox interactions

Regulation of Hox gene expression during anteroposterior patterning involves collinear Hox-Hox interactions

The Role of Histone Demethylases and DNA Methyltransferases in the Transcription Regulation of HOX Genes in PML-RARa+ AML Patients

Homeobox genes (HOX) encode transcription factors that are frequently deregulated in leukemias. Our previous results showed that HOX gene expression differs among genetically characterized subtypes of pediatric acute myeloid leukemia (AML). Specifically, PML-RARa positive AML patients have overall lowest HOX gene expression which positively correlates with expression of histone 3 lysine 27 (H3K27) demethylases - JMJD3 and UTX and negatively with the expression of DNA methyltransferases - DNMT3a and DNMT3b. Interestingly, JMJD3 was already shown to be a direct target of PML-RARa protein (Martens, JH et al, 2010, Cancer Cell). From these findings we postulated a hypothesis that reduced levels of HOX genes in PML-RARa positive AML are a consequence of suppressed expression of histone demethylases resulting in increased H3K27 methylation and/or of elevated levels of DNMTs leading to de novoDNA methylation. We studied the role of histone demethylases and DNMTs in the regulation of HOX gene expression and the effect of treatment in PML-RARa positive cell lines (NB4 and ATRA-resistant clones NB4-LR2 and NB4-MR2).We treated NB4 cell line by all-trans retinoic acid (ATRA; 1uM), which was described to release the differentiation block caused by the presence of PML-RARa and to degrade the fusion protein. We observed that expression of particular HOX genes (HOXA1, HOXA3, HOXA4, HOXA5, HOXA7, HOXB4, HOXB6) measured by qPCR was significantly increased after ATRA treatment. While the level of JMJD3 was significantly increased upon ATRA treatment as well, the expression of UTX did not change. Furthermore, we detected significantly reduced expression of DNMT3b gene. To exclude a non-specific effect of ATRA, independent of PML-RARa, we used resistant clones LR2 and MR2 bearing mutations in retinoic acid-binding domain. HOX gene expression together with JMJD3, UTX and DNMT3b expression did not change upon ATRA treatment. These results confirm the PML-RARa-dependent regulation of HOX genes. To test the role of JMJD3 in the HOX gene expression regulation, we cultured NB4 cells with a specific inhibitor of histone demethylases, GSK-J4 (1 uM, 10 uM), in the presence of ATRA. The co-treatment caused significant decrease in the expression of studied HOX genes (HOXA1, HOXA3, HOXA5, HOXA7, HOXA10, HOXB4, HOXB6) in comparison to ATRA alone which supports the role of JMJD3 in the transcription regulation. Further, we performed chromatin immunoprecipitation (ChIP) to investigate if the changes of HOX gene expression upon ATRA and GSK-J4 treatment would correspond with changes of histone code on HOX gene promoter regions. ATRA treatment caused reduction of repressive histone mark (H3K27me3) on particular HOX gene promoters (HOXA1, HOXA3, HOXA5, HOXA7), by contrast, combinational treatment of ATRA and GSK-J4 reversed this effect. Accordingly, we detected that ATRA/GSK-J4 co-treatment reduced active histone mark H3K4me2.Next we were interested if JMJD3 inhibition would interfere with the differentiation effect of ATRA. As shown previously, ATRA treatment alone caused differentiation of NB4 cell line whereas the combination with GSK-J4 did not reduce the effect. Interestingly, in addition to differentiation it led cells to apoptosis. Combination of drugs (ATRA - 1uM, GSK-J4 - 1, 2, 5uM) increased significantly the percentage of dead cells in comparison to ATRA or GSK treatment alone (GSK-J4 alone vs in combination with ATRA, 1uM - 1.8 fold, 2uM - 2.2 fold, 5 uM - 2.3 fold increase). Next we measured apoptosis in resistant clones LR2 and MR2. In both cases the highest concentration used of GSK-J4 (5uM) in combination with ATRA caused significant increase of dead cells as well (LR2 - 2.1 fold, MR2 - 2.0 fold increase).Our results indicate that JMJD3 is responsible for the regulation of HOX gene expression in PML-RARa positive leukemia since changes of HOX gene expression correspond with histone modifications on the regions of HOX gene promoters. We assume that DNA methylation driven by DNMT3b can also participate in this process. Moreover, our findings demonstrate potential therapeutic implications of GSK-J4 inhibitor in combination with ATRA in patients with acute promyelocytic leukemia who are not responsive to ATRA monotherapy.Supported by P304/12/2214 and GAUK 196616 DisclosuresNo relevant conflicts of interest to declare.

MIR142 Loss-of-Function Mutations Promote Leukemogenesis through Derepression of ASH1L Resulting in Increased HOX Gene Expression

Mutations of MIR142 have been identified in approximately 2% of de novo AML and in 20% of diffuse large B cell lymphoma (DLBCL). In AML, all of the mutations in MIR142 localize to the seed sequence of miRNA-142-3p, which is crucial in determining its target specificity. Previously, we showed that these mutations disrupt both miRNA-142-3p and miRNA-142-5p function, suggesting that loss of MIR142 plays a role in leukemic transformation (Yao et al, ASH abstract 1254, 2015). To test this hypothesis, we first characterized hematopoiesis in Mir142-/-mice. We previously reported that loss of Mir142 results in an expansion of myeloid progenitors with impaired erythropoiesis and lymphopoiesis (Yao, ASH abstract 1254, 2015). We now extend these analyses to investigate how loss of Mir142 promotes leukemic transformation.In the TCGA AML cohort, all 4 cases with somatic mutations in MIR142 also harbored mutations in either IDH1 or IDH2. To assess the functional importance of this association, we transduced wild-type or Mir142-/-hematopoietic stem/progenitor cells (HSPCs) with a retrovirus expressing the canonical IDH2 mutation, R172K. These cells were transplanted into lethally irradiated recipients and a tumor watch established. Loss of Mir142 alone was associated with mild splenomegaly, anemia, and leukopenia, but it was not sufficient to induce AML. Consistent with a prior report (Sasaki et al, Nature 2012), expression of IDH2 R172K alone induced a myeloproliferative disorder (MPD) characterized by increased myeloid cells, anemia, and splenomegaly. Concomitant loss of Mir142 did not affect the latency or penetrance of this MPD. However, the MPD in the double mutant mice was characterized by an increased percentage of CD34+ Gr1+ myeloblasts in the bone marrow and spleen plus a more severe anemia. To assess leukemia-initiating activity, we transplanted one million splenic cells into secondary recipients. Whereas IDH2 R172K alone cells rarely engrafted, Mir142-/- x IDH2 R172K cells efficiently engrafted and produced an MPD-like phenotype. These data suggest that loss of function mutations in MIR142 cooperate with IDH1/2 mutations to induce AML, possibly by increasing leukemic cell self-renewal.We examined several putative miR-142 target genes, eventually focusing on ASH1L. ASH1L is a member of the trithorax family of histone methyltransferases that has been recently implicated in MLL-associated leukemogenesis. The 3' UTR of ASH1L contains 4 putative binding sites for miRNA-142-3p, suggesting that this miRNA is critical in its post-transcriptional regulation. Indeed, in a luciferase assay with the ASH1L 3' UTR, MIR142 overexpression decreased translation by 80 percent. Consequently, Ash1l protein levels were 3 fold higher in Mir142-/- mice bone marrow compared to control mice. Since ASH1L is a key regulator of HOX gene expression, we examined HoxA9 and HoxA10 expression in Mir142-/- hematopoietic progenitor subsets. While HoxA9 and HoxA10 expression were not different in hematopoietic stem cells, they were markedly upregulated in myeloid progenitors. For example, in granulocyte-macrophage progenitors (GMPs), HoxA9 and HoxA10 expression were increased 2.86-fold and 34.4-fold, respectively in Mir142-/- versus control cells. Likewise, in megakaryocyte-erythroid progenitors (MEPs), HoxA9 and HoxA10 expression were increased 5.3-fold and 21.4-fold. Dysregulated HoxA9 and HoxA10 expression have been implicated in enhanced self-renewal capacity, and HoxA9 overexpression has been shown to cooperate with mutant IDH1 to induce AML in mice (Chaturvedi et al, Blood 2013). Collectively, these data suggest a model in which MIR142 mutations contribute to leukemogenesis by derepressing ASH1L expression, which, in turn, increases expression of HoxA9/10 and enhances self-renewal. Inhibitors targeting ASH1L may have therapeutic benefit in AML characterized by increased HOX gene expression. DisclosuresNo relevant conflicts of interest to declare.

The Histone Methyltransferase Gene Absent, Small, or Homeotic Discs-1 Like Is Required for Normal Hox Gene Expression and Fertility in Mice.

Chromatin remodeling influences gene expression in developing and adult organisms. Active and repressive marks of histone methylation dictate the embryonic expression boundaries of developmentally regulated genes, including the Hox gene cluster. Drosophila ash1 (absent, small or homeotic discs 1) gene encodes a histone methyltransferase essential for regulation of Hox gene expression that interacts genetically with other members of the trithorax group (TrxG). While mammalian members of the mixed lineage leukemia (Mll) family of TrxG genes have roles in regulation of Hox gene expression, little is known about the expression and function of the mammalian ortholog of the Drosophila ash1 gene, Ash1-like (Ash1l). Here we report the expression of mouse Ash1l gene in specific structures within various organs and provide evidence that reduced Ash1l expression has tissue-specific effects on mammalian development and adult homeostasis. Mutants exhibit partially penetrant postnatal lethality and failure to thrive. Surviving mutants have growth insufficiency, skeletal transformations, and infertility associated with developmental defects in both male and female reproductive organs. Specifically, expression of Hoxa11 and Hoxd10 are altered in the epididymis of Ash1l mutant males and Hoxa10 is reduced in the uterus of Ash1l mutant females. In summary, we show that the histone methyltransferase Ash1l is important for the development and function of several tissues and for proper expression of homeotic genes in mammals.

F9 EC 세포에서 레티노산에 의해 유도되는 Hoxc 유전자의 발현에 히스톤 메틸화가 미치는 영향

Department of Anatomy, Embryology Laboratory, Brain Korea 21 Pl us project for Medical Science, Yonsei University College of Medicine, Seoul 120-752, KoreaReceived December 1, 2014/Revised March 11, 2015/Accepted March 12, 2015Hox genes encode a highly conserved family of homeodomain-containing transcription factors control-ling vertebrate pattern formation along the anteroposterior body axis during embryogenesis. Retinoic acid (RA) is a key morphogen in embryogenesis and a critical regulator of both adult and embryonic cellular activity. Specifically, RA regulates Hox gene expression in mouse- or human-derived embry-onic carcinoma (EC) cells. Histone modification has been reported to play a pivotal role in the process of RA-induced gene expression and cell differentiation. As histone modification is thought to play an essential role in RA-induced Hox gene expression, we examined RA-induced initiation of collinear ex-pression of Hox genes and the corresponding histone modifications in F9 murine embryonic ter-atocarcinoma (EC) cells. Hox expression patterns and histone modifications were analyzed by semi- quantitative RT-PCR, RNA-sequencing, and chromatin immuno-precipitation (ChIP)-PCR analyses. The Hoxc4 gene (D0) was initiated earlier than the Hoxc5 to –c10 genes (D3) upon RA treatment (day 0 [D0], day 1 [D1], and day 3 [D3]). The Hox nonexpressing D0 sample had a strong repressive marker, H3K27me3, than the D1 and D3 samples. In the D1 and D3 samples, reduced enrichment of the H3K27me3 marker was observed in the whole cluster. The active H3K4me3 marker was closely asso-ciated with the collinear expression of Hoxc genes. Thus, the Hoxc4 gene (D1) and all Hoxc genes (D3) expressed H3K4me3 upon transcription activation. In conclusion, these data indicated that removing H3K27me3 and acquiring H3K4me3 regulated RA-induced Hoxc gene collinearity in F9 cells. Keywords :

The Role of Histone Demethylases in the Transcription Regulation of HOX Genes in PML-RARa+ AML Patients

Homeobox genes (HOX) encode transcription factors that are frequently deregulated in leukemias. Our previous findings described that HOX gene expression differs among genetically characterized subtypes of pediatric AML with PML-RARa+ patients having the lowest overall HOX gene expression. We observed that HOX gene expression positively correlated with expression of histone 3 lysine 27 (H3K27) demethylases JMJD3 and UTX and negatively with DNA methyltransferase DNMT3b. Interestingly, it has been shown that JMJD3 is a direct target of PML-RARa protein (Martens, JH et al, 2010, Cancer Cell). These findings led us to postulate the hypothesis that reduced levels of HOX genes in PML-RARa+ AML can be caused by the suppressed expression of histone demethylases, such as JMJD3 and UTX, resulting in increased H3K27 methylation and transcription inhibition.We chose PML-RARa+ NB4 cell line to study the role of PML-RARa fusion gene in the regulation of HOX gene expression. To inhibit the effect of PML-RARa we used all-trans retinoic acid (ATRA; 1 uM, 10 uM) which was described to release the block caused by this fusion protein. Expression of particular HOX genes (e.g., HOXA1, HOXA3, HOXA5, HOXA7) together with that of JMJD3 and UTX assessed by qPCR was significantly elevated after ATRA treatment, while gene expression of DNMT3b was decreased.To test whether the reduction in HOX gene expression is directly related to the levels of JMJD3 and UTX, we cultured NB4 cells with a specific inhibitor of these histone demethylases, GSK-J4 (1 uM, 10 uM), in combination with ATRA. This co-treatment led to inhibition of JMJD3 and UTX proteins, followed by significant reduction of HOX genes expression (e.g., HOXA1, HOXA3, HOXA5, HOXA7). This result supports our hypothesis that HOX genes expression is directly related to JMJD3/UTX activity.To determine the effect of ATRA and GSK-J4 on histone marks we have isolated histones by acid extraction and detected the levels of histones by western blot in NB4 ATRA or GSK-J4/ATRA treated cells. We observed that the level of repressive histone methylation mark (trimethylated H3K27; H3K27me3) was decreased after ATRA treatment (activation of JMJD3/UTX) and increased after GSK-J4/ATRA co-treatment (inhibition of JMJD3/UTX). The opposite effect was observed in active histone methylation marks where di- and tri-methylated H3K4 (H3K4me2, H3K4me3) increased after ATRA treatment and decreased after GSK-J4/ATRA co-treatment. H3K9 dimethylated (another repressive histone methylation mark) levels did not change.Next, to investigate the histone code directly in particular HOX genes regions we performed chromatin immunoprecipitation (ChIP) assays. We studied the presence of H3K27me3 and H3K4me2 in 5´UTR genomic region of particular HOX genes (HOXA1, HOXA2, HOXA3, HOXA5, HOXA7) in cells treated with ATRA alone or in the combination with GSK-J4. Preliminary results showed reduction in repressive marks (H3K27me3) upon ATRA treatment, whereas addition of GSK-J4 prevented this decrease. Accordingly, we observed that ATRA/GSK-J4 co-treatment reduced active histone mark H3K4me2.To evaluate the role of DNA methylation in observed expression changes after ATRA treatment we performed bisulfite sequencing of particular promoter sites of HOX genes (e.g., HOXA7, HOXA5). Although we detected decreased DNMT3b gene expression after ATRA treatment there was no change in DNA methylation of CpGs in studied regions.Our results demonstrate that changes in chromatin activity correspond with changes in HOX gene expression. Moreover, ChIP data show direct binding of the modified histones and HOX 5´UTR sites. Our data implicate histone demethylases in regulation of HOX gene expression in PML-RARa+ leukemic blasts. DNA methylation in these particular HOX genes is not involved in the regulation. Elucidating the mechanism of regulation of HOX genes expression can help to understand their role in the leukemogenic process.Supported by GACR P304/12/2214 and GAUK 568213. DisclosuresNo relevant conflicts of interest to declare.

Nucleoporin Nup98 Associates with Trx/MLL and NSL Histone-Modifying Complexes and Regulates Hox Gene Expression

The nuclear pore complex is a transport channel embedded in the nuclear envelope and made up of 30 different components termed nucleoporins (Nups). In addition to their classical role in transport, a subset of Nups has a conserved role in the regulation of transcription via direct binding to chromatin. The molecular details of this function remain obscure, and it is unknown how metazoan Nups are recruited to their chromatin locations or what transcription steps they regulate. Here, we demonstrate genome-wide and physical association between Nup98 and histone-modifying complexes MBD-R2/NSL [corrected] and Trx/MLL. Importantly, we identify a requirement for MBD-R2 in recruitment of Nup98 to many of its genomic target sites. Consistent with its interaction with the Trx/MLL complex, Nup98 is shown to be necessary for Hox gene expression in developing fly tissues. These findings introduce roles of Nup98 in epigenetic regulation that may underlie the basis of oncogenicity of Nup98 fusions in leukemia.

Regulation of HOX gene expression by AF10-mediated conversion of H3K79me1 to H3K79me2

Regulation of HOX gene expression by AF10-mediated conversion of H3K79me1 to H3K79me2

Multiple transcription factors directly regulate Hox gene lin-39 expression in ventral hypodermal cells of the C. elegans embryo and larva, including the hypodermal fate regulators LIN-26 and ELT-6.

BackgroundHox genes encode master regulators of regional fate specification during early metazoan development. Much is known about the initiation and regulation of Hox gene expression in Drosophila and vertebrates, but less is known in the non-arthropod invertebrate model system, C. elegans. The C. elegans Hox gene lin-39 is required for correct fate specification in the midbody region, including the Vulval Precursor Cells (VPCs). To better understand lin-39 regulation and function, we aimed to identify transcription factors necessary for lin-39 expression in the VPCs, and in particular sought factors that initiate lin-39 expression in the embryo.ResultsWe used the yeast one-hybrid (Y1H) method to screen for factors that bound to 13 fragments from the lin-39 region: twelve fragments contained sequences conserved between C. elegans and two other nematode species, while one fragment was known to drive reporter gene expression in the early embryo in cells that generate the VPCs. Sixteen transcription factors that bind to eight lin-39 genomic fragments were identified in yeast, and we characterized several factors by verifying their physical interactions in vitro, and showing that reduction of their function leads to alterations in lin-39 levels and lin-39::GFP reporter expression in vivo. Three factors, the orphan nuclear hormone receptor NHR-43, the hypodermal fate regulator LIN-26, and the GATA factor ELT-6 positively regulate lin-39 expression in the embryonic precursors to the VPCs. In particular, ELT-6 interacts with an enhancer that drives GFP expression in the early embryo, and the ELT-6 site we identified is necessary for proper embryonic expression. These three factors, along with the factors ZTF-17, BED-3 and TBX-9, also positively regulate lin-39 expression in the larval VPCs.ConclusionsThese results significantly expand the number of factors known to directly bind and regulate lin-39 expression, identify the first factors required for lin-39 expression in the embryo, and hint at a positive feedback mechanism involving GATA factors that maintains lin-39 expression in the vulval lineage. This work indicates that, as in other organisms, the regulation of Hox gene expression in C. elegans is complicated, redundant and robust.

A key role for EZH2 in epigenetic silencing of HOX genes in mantle cell lymphoma

The chromatin modifier EZH2 is overexpressed and associated with inferior outcome in mantle cell lymphoma (MCL). Recently, we demonstrated preferential DNA methylation of HOX genes in MCL compared with chronic lymphocytic leukemia (CLL), despite these genes not being expressed in either entity. Since EZH2 has been shown to regulate HOX gene expression, to gain further insight into its possible role in differential silencing of HOX genes in MCL vs. CLL, we performed detailed epigenetic characterization using representative cell lines and primary samples. We observed significant overexpression of EZH2 in MCL vs. CLL. Chromatin immune precipitation (ChIP) assays revealed that EZH2 catalyzed repressive H3 lysine 27 trimethylation (H3K27me3), which was sufficient to silence HOX genes in CLL, whereas in MCL H3K27me3 is accompanied by DNA methylation for a more stable repression. More importantly, hypermethylation of the HOX genes in MCL resulted from EZH2 overexpression and subsequent recruitment of the DNA methylation machinery onto HOX gene promoters. The importance of EZH2 upregulation in this process was further underscored by siRNA transfection and EZH2 inhibitor experiments. Altogether, these observations implicate EZH2 in the long-term silencing of HOX genes in MCL, and allude to its potential as a therapeutic target with clinical impact.

Leukemic Pattern Of HOX Gene Expression Is Driven By Genetic Aberrations Through Epigenetic Modifiers

IntroductionHomeobox (HOX) genes encode transcription factors crucial in embryogenesis. They are often dysregulated in malignancies including leukemias. The aberrant HOX gene expression and its regulation in leukemic cells is neither completely described nor understood. AimsOur main aim was to determine whether the leukemic HOX gene expression pattern is driven by differentiation stage of hematopoietic cells or determined de novo during the process of malignant transformation. Consequentially, we aimed to study the role epigenetic modifiers in regulation of HOX gene expression in normal and malignant hematopoiesis. MethodsThe expression pattern of HOX genes (cluster of HOX A and B) and epigenetic modifiers (DNMT1, DNMT3a, DNMT3b, EZH2, BMI-1, MLL, JMJD3, UTX) was assessed by qPCR in 8 FACS-sorted subpopulations of healthy BM representing stages of myeloid differentiation (each sample representing a pool of cells sorted from five individuals). The leukemic expression pattern of these genes was analyzed in diagnostic BM samples of childhood AML patients with typical genotypic and morphological (FAB classification) characteristics (N=46). In vitro experiments were performed using NB4 cell line. ResultsAs expected HOX genes were gradually downregulated during normal differentiation of granulocytic and monocytic lineages (assessed in four consecutive differentiation stages for each lineage). In AML samples, HOX gene expression patterns differed significantly among morphological subtypes. However, HOX gene expression did not correlate among subtypes of AML and their physiologically differentiated counterparts.Interestingly, unsupervised hierarchical clustering (HCA) divided AML patients into four main clusters characterized by the presence of prevalent gene rearrangement (PML-RARa, AML1-ETO, MLL rearrangements and NK-AML). The presence of PML/RARa rearrangement was strongly associated with the lowest expression of both HOXA and HOXB clusters, while the other groups had more variable expression of HOX genes. Moreover, the effect of genetic aberrations on HOX gene expression was clearly apparent within AML M2 and M4 subtypes, where AML1/ETO+ or CBFb/MYH11+ patients had significantly lower expression of HOX genes compared to patients with the same FAB classification but without the rearrangements.The expression pattern of epigenetic modifiers in sorted subpopulations of healthy BM followed their expected role in transcriptional regulation during differentiation. However, there was no relation of this pattern to HOX gene expression. On the contrary, in AML samples, the expression levels of epigenetic modifiers clearly correlated with expression profile of HOX genes. These results were supported by unsupervised HCA based on the expression of epigenetic modifiers that showed upregulation of histon demethylases JMJD3 and UTX together with downregulation of DNMT3b in concordance with high levels of HOX genes. Negative correlation between JMJD3 and DNMT3b expression was observed in all leukemic samples (p=0.03); most apparently in PML/RARa+ patients.Therefore we further studied the impact of genetic aberrations on the epigenetic regulation of HOX gene expression in vitrowith PML-RARa+ cell line. Treatment of NB4 cells with ATRA (8, 24hours, 1uM, 10uM) increased the levels of particular HOX genes (HOXA5, A7, B4, B7; FCA=2.8; 1.7; 4; 4 respectively) as well as JMJD3 (FCA=3) and UTX (FCA=1.6). Concordantly, the expression of DNMT3b (FCA=5) was downregulated.The hypothetical driving effect of PML-RARa on de novo determination of leukemic HOX gene expression is further supported by our Results. PML-RARa+ patients had the lowest HOX gene expression regardless of their FLT3/ITD status – previously shown to upregulate strongly HOX genes expression. ConclusionWe conclude that the leukemic expression pattern of HOX genes does not reflect the differentiation stages of malignant cells. Our data also demonstrate different contribution of epigenetic modifiers to the HOX gene expression in healthy and malignant hematopoiesis. Moreover, HCA and expression data together with the results of in vitro experiments suggest that the specific molecular aberrations (as exemplified by PML-RARa) participate in regulation of leukemic HOX gene expression through epigenetic changes.Supported by GACR P304/12/2214, GAUK 568213, 00064203. Disclosures:No relevant conflicts of interest to declare.