H2B K120 Research Articles

GSE1 links the HDAC1/CoREST co-repressor complex to DNA damage.

Post-translational modifications of histones are important regulators of the DNA damage response (DDR). By using affinity purification mass spectrometry (AP-MS) we discovered that genetic suppressor element 1 (GSE1) forms a complex with the HDAC1/CoREST deacetylase/demethylase co-repressor complex. In-depth phosphorylome analysis revealed that loss of GSE1 results in impaired DDR, ATR signalling and γH2AX formation upon DNA damage induction. Altered profiles of ATR target serine-glutamine motifs (SQ) on DDR-related hallmark proteins point to a defect in DNA damage sensing. In addition, GSE1 knock-out cells show hampered DNA damage-induced phosphorylation on SQ motifs of regulators of histone post-translational modifications, suggesting altered histone modification. While loss of GSE1 does not affect the histone deacetylation activity of CoREST, GSE1 appears to be essential for binding of the deubiquitinase USP22 to CoREST and for the deubiquitination of H2B K120 in response to DNA damage. The combination of deacetylase, demethylase, and deubiquitinase activity makes the USP22-GSE1-CoREST subcomplex a multi-enzymatic eraser that seems to play an important role during DDR. Since GSE1 has been previously associated with cancer progression and survival our findings are potentially of high medical relevance.

Histone methyltransferase Dot1L recruits O-GlcNAc transferase to target chromatin sites to regulate histone O-GlcNAcylation

O-GlcNAc transferase (OGT) is the distinctive enzyme responsible for catalyzing O-GlcNAc addition to the serine or threonine residues of thousands of cytoplasmic and nuclear proteins involved in such basic cellular processes as DNA damage repair, RNA splicing, and transcription preinitiation and initiation complex assembly. However, the molecular mechanism by which OGT regulates gene transcription remains elusive. Using proximity labeling-based mass spectrometry, here, we searched for functional partners of OGT and identified interacting protein Dot1L, a conserved and unique histone methyltransferase known to mediate histone H3 Lys79 methylation, which is required for gene transcription, DNA damage repair, cell proliferation, and embryo development. Although this specific interaction with OGT does not regulate the enzymatic activity of Dot1L, we show that it does facilitate OGT-dependent histone O-GlcNAcylation. Moreover, we demonstrate that OGT associates with Dot1L at transcription start sites and that depleting Dot1L decreases OGT associated with chromatin globally. Notably, we also show that downregulation of Dot1L reduces the levels of histone H2B S112 O-GlcNAcylation and histone H2B K120 ubiquitination in vivo, which are associated with gene transcription regulation. Taken together, these results reveal that O-GlcNAcylation of chromatin is dependent on Dot1L.

Chemical Catalysis Intervening to Histone Epigenetics.

Life emerges from complicated and sophisticated chemical networks comprising numerous biomolecules (e.g., nucleic acids, proteins, sugars, and lipids) and chemical reactions catalyzed by enzymes. Dysregulation of these chemical networks is linked to the emergence of diseases. Our research goal is to develop abiotic chemical catalysts that can intervene into life's chemical networks by complementing, surrogating, or exceeding enzymes in living cells or multicellular organisms such as animals or plants. Mending dysregulated networks in pathological states by the chemical catalysts will lead to a new medicinal strategy, catalysis medicine. This research direction will also advance catalysis science, because highly active and selective chemical catalysts must be developed to promote the intended reactions in a complex mixture of life in aqueous solution at body temperature.Epigenetics exists at the crossroads of chemistry, biology, and medicine and is a suitable field to pursue this idea. Post-translational modifications (PTMs) of histones epigenetically regulate chromatin functions and gene transcription and are intimately related to various diseases. Investigating the functions and cross-talk of histone PTMs is crucial for mechanistic elucidation of diseases and their treatments. We launched a program to develop chemical catalysts enabling endogenous histone modifications in living cells without relying on enzymes. We reported two types of chemical catalyst systems so far for synthetic histone acylation. The first system comprised a DNA-binding oligo-4-dimethylaminopyridine (DMAP) catalyst and a phenyl ester acyl donor, PAc-gly. This system promoted histone hyperacetylation in Xenopus laevis sperm chromatin. Using the thus-synthesized hyperacetylated sperm chromatin, we found a novel relationship between histone acetylation and DNA replication. The second system involved a histone-binding catalyst, LANA-DSH, composed of a catalytic motif (DSH) and a histone-binding peptide ligand (LANA), and thioester acyl donors, including endogenous acyl-CoA. This system regioselectively (i.e., selectively to a lysine residue at a specific position) acylated lysine 120 of histone H2B (H2BK120), a lysine residue proximal to the DSH motif defined by binding of the LANA ligand to a nucleosome substrate. This catalyst system was optimized to achieve H2BK120-selective acetylation in living cells without genetic manipulation. The synthetically introduced H2BK120Ac inhibited enzyme-catalyzed ubiquitination at the same lysine residue, acting as a protecting group. H2BK120Ub is a mark recognized by methyltransferase that plays an essential role in mixed-lineage leukemia (MLL)-rearranged leukemia, suggesting the potential of the catalyst system as an epigenetic tool and a cancer therapy. We also discuss the prospects of chemical catalyst-promoted synthetic epigenetics for future PTM studies and therapeutic uses.

Linc-RA1 inhibits autophagy and promotes radioresistance by preventing H2Bub1/USP44 combination in glioma cells

Radiotherapy is one of the standard treatments for glioma patients; however, its clinical efficacy is limited by radioresistance. We identified a mechanism of such resistance mediated by linc-RA1 (radioresistance-associated long intergenic noncoding RNA 1). Linc-RA1 was upregulated in radioresistant glioma cells and glioma tissue samples, compared with radiosensitive cells and nontumor tissues. Linc-RA1 was associated with inferior overall survival and advanced clinical stage of glioma. Linc-RA1 promoted glioma radioresistance in vitro and in vivo. Mechanistically, linc-RA1 stabilized the level of H2B K120 monoubiquitination (H2Bub1) by combining with H2B and inhibiting the interaction between H2Bub1 and ubiquitin-specific protease 44 (USP44), which inhibited autophagy, thus contributing to glioma radioresistance. These results reveal that linc-RA1-mediated autophagy is a key mechanism of radioresistance and is an actionable target for improving radiotherapy efficacy in patients with glioma.

Mechanism of Cross-talk between H2B Ubiquitination and H3 Methylation by Dot1L

Methylation of histone H3 K79 by Dot1L is a hallmark of actively transcribed genes that depends on monoubiquitination of H2B K120 (H2B-Ub) and is an example of histone modification cross-talk that is conserved from yeast to humans. We report here cryo-EM structures of Dot1L bound to ubiquitinated nucleosome that show how H2B-Ub stimulates Dot1L activity and reveal a role for the histone H4 tail in positioning Dot1L. We find that contacts mediated by Dot1L and the H4 tail induce a conformational change in the globular core of histone H3 thatreorients K79 from an inaccessible position, thus enabling this side chain to insert into the active site in a position primed for catalysis. Ourstudy provides a comprehensive mechanism ofcross-talk between histone ubiquitination and methylation and reveals structural plasticity in histones that makes it possible for histone-modifying enzymes to access residues within the nucleosome core.

An IGF2BP3-Cdk9 Pathway Governs the Human Fetal-Adult Megakaryocyte Transition

Hematopoietic transitions that accompany fetal development, for example erythroid globin chain switching, play important roles in normal physiology and disease development. In the megakaryocyte lineage, human fetal progenitors show impaired execution of the morphogenesis program of enlargement, polyploidization, and proplatelet formation. Although these defects decline with gestational stage, they remain sufficiently severe at birth to predispose newborns to thrombocytopenia. These defects also contribute to inferior platelet recovery after cord blood stem cell transplantation and to inefficient platelet production by megakaryocytes (Mk) derived from pluripotent stem cells. In this study, comparison of neonatal versus adult human progenitors identified a blockade in the specialized positive transcription elongation factor b (P-TEFb) activation mechanism known to drive adult Mk morphogenesis. A central feature of this pathway is known to involve sustained high amplitude activation of the P-TEFb kinase (Cdk9/cyclin T1). This cascade is initiated by downregulation of core components of the repressive complex of P-TEFb: the 7SK snRNP consisting of the 7SK small nuclear RNA (7SK) and its stabilizing factors MePCE and LARP7. The resulting high amplitude activation of P-TEFb drives multiple features of Mk differentiation: induction of cytoskeletal morphogenetic factors (ACTN1, FLNA, MKL1, HIC5), silencing of erythroid genes, promotion of histone H2B K120 monoubiquitiniation (H2BUb1), and phosphorylation of Spt5 at T806 (pSpt5 T806). Critical, rate-limiting steps triggering this pathway comprise MePCE proteolysis by calpain 2 and downregulation of LARP7, both resulting in destabilization of 7SK.In comparison to Mk derived from adult peripheral blood stem cells (adult Mk), Mk derived from umbilical cord blood stem cells (neonatal Mk) showed evidence of decreased P-TEFb activation with decreases in: 1) the expression of the cytoskeletal morphogenetic factors, 2) global H2BUb1, and 3) pSpt5 T806. In addition, neonatal Mk retained expression of the erythroid marker glycophorin A (GPA). Surprisingly, neonatal Mk failed to downregulate 7SK despite the downregulation of its stabilizers MePCE and LARP7, suggesting the existence of alternative 7SK stabilizing factor/s unique to neonatal Mk. Our screening identified the oncofetal RNA-binding protein IGF2BP3 to be expressed in neonatal but not adult Mk, and ectopic expression of IGF2BP3 in adult Mk conferred neonatal phenotypic features including reduction in size, increased proliferation and leaky erythroid antigen expression. Immunoprecipitation and glycerol gradient studies indicated the participation of IGF2BP3 in the 7SK snRNP complex. Mining of endogenous IGF2BP3 iCLIP data indicated that 7SK is one of the top direct targets of IGF2BP3 and further mapped binding to the 7SK fourth hairpin, a critical stability determinant.In loss of function studies, the knockdown of IGF2BP3 in neonatal Mk resulted in destabilization of 7SK and upregulation of the Mk morphogenetic cytoskeletal factors, as well as increased levels of H2BUb1, pSpt5 T806, and hyperphosphorylated RNA Pol II. Phenotypically, the knockdown of IGF2BP3 in neonatal Mk elicited adult features including increased size, enhanced polyploidization, reduced proliferation and silencing of erythroid antigen expression. Collectively, these findings suggest that the block in P-TEFb activation in neonatal Mk results from ontogentic stage-specific expression of IGF2BP3 which prevents the 7SK destabilization normally associated with adult megakaryocytic P-TEFb activation. We also identified a pharmacologic approach to inhibit IGF2BP3 expression, through inhibition of bromodomain and extra- terminal (BET) proteins, which reproducibly promoted adult features in neonatal Mk including enlargement, inhibition of erythroid antigen expression, upregulation of morphogenetic cytoskeletal factors, and increased platelet formation in vitro. Enforced expression of IGF2BP3 in neonatal Mk significantly blunted the effects of BET inhibitors indicating the specificity of their action in downregulating IGF2BP3. These results identify IGF2BP3 as a human ontogenic masterswitch that restricts megakaryocyte development through modulating a lineage-specific P-TEFb activation mechanism, revealing new strategies toward enhancing platelet production. DisclosuresNo relevant conflicts of interest to declare.

Structural and biochemical analyses of monoubiquitinated human histones H2B and H4.

Monoubiquitination is a major histone post-translational modification. In humans, the histone H2B K120 and histone H4 K31 residues are monoubiquitinated and may form transcriptionally active chromatin. In this study, we reconstituted nucleosomes containing H2B monoubiquitinated at position 120 (H2Bub120) and/or H4 monoubiquitinated at position 31 (H4ub31). We found that the H2Bub120 and H4ub31 monoubiquitinations differently affect nucleosome stability: the H2Bub120 monoubiquitination enhances the H2A–H2B association with the nucleosome, while the H4ub31 monoubiquitination decreases the H3–H4 stability in the nucleosome, when compared with the unmodified nucleosome. The H2Bub120 and H4ub31 monoubiquitinations both antagonize the Mg2+-dependent compaction of a poly-nucleosome, suggesting that these monoubiquitinations maintain more relaxed conformations of chromatin. In the crystal structure, the H2Bub120 and H4ub31 monoubiquitinations do not change the structure of the nucleosome core particle and the ubiquitin molecules were flexibly disordered in the H2Bub120/H4ub31 nucleosome structure. These results revealed the differences and similarities of the H2Bub120 and H4ub31 monoubiquitinations at the mono- and poly-nucleosome levels and provide novel information to clarify the roles of monoubiquitination in chromatin.

Histone post-translational modifications in frontal cortex from human donors with Alzheimer’s disease

BackgroundAlzheimer’s disease (AD) is the sixth leading cause of death and the most costly disease in the US. Despite the enormous impact of AD, there are no treatments that delay onset or stop disease progression currently on the market. This is partly due to the complexity of the disease and the largely unknown pathogenesis of sporadic AD, which accounts for the vast majority of cases. Epigenetics has been implicated as a critical component to AD pathology and a potential “hot spot” for treatments. Histone post-translational modifications (PTMs) are a key element in epigenetic regulation of gene expression and are known to be associated with the pathology of numerous diseases. Investigation of histone PTMs can help elucidate AD pathology and identify targets for therapies.ResultsA multiple reaction monitoring mass spectrometry assay was used to measure changes in abundance of several histone PTMs in frontal cortex from human donors affected with AD (n = 6) and age-matched, normal donors (n = 6). Of the changes observed, notable decreases in methylation of H2B residue K108 by 25 % and H4 residue R55 by 35 % were measured and are likely associated with hydrogen bonding networks important for nucleosome stability. Additionally, a 91 % increase in ubiquitination of K120 on H2B was measured as well as an apparent loss in acetylation of the region near the N-terminus of H4. Our method of quantification was also determined to be precise and robust, signifying measured changes were representative of true biological differences between donors and sample groups.ConclusionWe are the first to report changes in methylation of H2B K108, methylation of H4 R55, and ubiquitination of H2B K120 in frontal cortex from human donors with AD. These notable PTM changes may be of great importance in elucidating the epigenetic mechanism of AD as it relates to disease pathology. Beyond the structural and functional impacts of the changes we have measured, the sites of altered PTMs may be used to identify enzymes responsible for their modulation, which could be used as prospective drug targets for highly specific AD therapies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12014-015-9098-1) contains supplementary material, which is available to authorized users.

The Distinctive Morphogenesis of Infantile Megakaryopoiesis Is Associated with Altered P-TEFb Regulation By the Oncofetal Factor IGF2BP3

Infantile (fetal/neonatal) megakaryocytes (Mk) differ from "adult" Mk in several important aspects. They are smaller, more proliferative, less polyploid and show leaky expression of erythroid genes. Their distinctive properties contribute to multiple disease states including neonatal thrombocytopenia, poor platelet recovery post umbilical cord blood (CB) transplantation, and acute Mk leukemias (AMkL). Their leukemic propensity is highlighted by the capacity of the AMkL oncoprotein GATA1s to transform infantile but not adult Mk. Despite their altered morphogenesis, infantile megakaryocytes retain most features of the adult differentiation and signaling program. Their principal signaling perturbation has been characterized as excessive responsiveness to thrombopoietin (Tpo) particularly with regard to the mTOR pathway. Thus Tpo agonists used to treat adult thrombocytopenia may not offer appropriate therapy for neonatal thrombocytopenia.We previously identified a signaling pathway that drives megakaryocyte morphogenesis and is disrupted by the GATA1s oncoprotein in AMkL (Elagib et al., Dev. Cell, 2013). A central feature of this pathway is the irreversible activation of the P-TEFb kinase (Cdk9/Cyclin T). This cascade is initiated by downregulation of core components of the repressive 7SK snRNP complex (MePCE, LARP7, 7SK snRNA). The resulting constitutive P-TEFb activation drives multiple features of Mk differentiation: induction of cytoskeletal morpho-genetic factors, silencing of erythroid genes, and promotion of histone H2B K120 monoubiquitiniation (H2BUb1). A critical, rate-limiting step triggering this pathway comprises MePCE proteolysis by calpain 2. GATA1s disrupts this pathway by preventing induction of calpain 2 by wild type GATA1.We now report that infantile Mk display intrinsic defects in the Mk P-TEFb activation pathway. In repeated experiments, human CB Mk failed to upregulate P-TEFb-dependent cytoskeletal factors, exhibited global deficiency in H2BUb1, and incompletely silenced erythroid antigen expression. Their defective P-TEFb activation resulted from an inability to downregulate 7SK snRNA, despite downregulation of the key 7SK stabilizers MePCE and LARP7. The inexplicable stabilization of 7SK in CB Mk argues for the existence of an alternative, infantile 7SK snRNP complex refractory to activation by calpain. Accordingly, screening studies identified candidate 7SK binding factors preferentially expressed in CB as opposed to adult progenitors. Among these factors, the RNA binding protein IGF2BP3 showed high abundance in CB Mk but complete absence from adult peripheral blood-derived (PB) Mk. Furthermore, immunoprecipitation studies consistently showed interaction of endogenous IGF2BP3 and HEXIM1 in K562 cells. HEXIM1 is the 7SK snRNP component that mediates repression of P-TEFb. Immunoprecipitation of epitope-tagged IGF2BP3 from HEK293 cells consistently identified an association with endogenous 7SK snRNA. In addition, enforced expression of IGF2BP3 in HEK293 cells, to levels seen in CB Mk, shifted the fractionation pattern of HEXIM1 on glycerol gradient sedimentation. Notably, a similar difference in HEXIM1 fractionation was seen when comparing CB and adult Mk by glycerol gradient sedimentation. Thus, IGF2BP3 represents a fetal/neonatal factor that reconfigures the composition and/or conformation of the 7SK snRNP, potentially altering regulation of P-TEFb. Contribution of IGF2BP3 to the infantile Mk phenotype was supported by experiments in which shRNA-mediated knockdown in CB Mk consistently enhanced enlargement, polyploidization, growth arrest, and erythroid silencing. Conversely, enforced expression of IGF2BP3 in adult Mk inhibited their enlargement, polyploidization, and growth arrest. Our results thus implicate IGF2BP3 as a key contributor to the infantile Mk phenotype, interfering with morphogenesis by stabilizing 7SK and thwarting irreversible P-TEFb activation. In light of our prior published results on the inhibitory effects of GATA1s in Mk morphogenesis (Elagib et al., Dev. Cell, 2013), our current findings highlight P-TEFb regulation as a convergence point for oncogenic stimuli during megakaryopoiesis. DisclosuresNo relevant conflicts of interest to declare.

MacroH2A1.1 and PARP-1 cooperate to regulate transcription by promoting CBP-mediated H2B acetylation

The histone variant macroH2A1 regulates gene expression important for differentiation, stem cell reprogramming and tumor suppression. Here, we demonstrate that in primary human cells, macroH2A1 participates in two physically and functionally distinct types of chromatin either marked by H3K27me3 or nine histone acetylations. Using RNA-seq, we found that macroH2A1-regulated genes, which have roles in cancer progression, are specifically found in macroH2A1-containing acetylated chromatin. Of the two macroH2A1 variants, macroH2A1.1 and macroH2A1.2, the former is suppressed in cancer and can interact with PARP-generated poly(ADP-ribose). Through the recruitment of PARP-1, macroH2A1.1 promotes the CBP-mediated acetylation of H2B K12 and K120 which either positively or negatively regulates the expression of macroH2A1-target genes. While macroH2A1-regulated H2B acetylation is a common feature of primary cells, this regulation is typically lost in cancer cells. Consequently, our results provide important insight into macroH2A1.1’s role in cancer suppression.

Abstract 5156: The oncogenic polycomb histone methyltransferase EZH2 methylates lysine 120 on histone H2B and competes ubiquitination in human cancer

Abstract The histone methyltransferase EZH2 is known to be a polycomb protein homologus to Drosophila enhancer of zeste and catalyzes the addition of methyl groups to histone H3 at lysine 27 (H3K27). We previously reported that EZH2 was overexpressed in various types of cancer and plays a crucial role in the cell cycle regulation of cancer cells. In this study, we demonstrated that EZH2 has the function to mono-methylated lysine 120 on histone H2B (H2BK120). EZH2-dependent H2BK120 methylation in cancer cells was confirmed with an H2BK120 methylation-specific antibody. Overexpression of EZH2 significantly attenuated the ubiquitination of H2BK120, a key post-translational modification of histones for transcriptional regulation. Concordantly, knockdown of EZH2 increased the ubiquitination level of H2BK120, suggesting that the methylation of H2BK120 by EZH2 may competitively inhibit the ubiquitination of H2BK120. Subsequent ChIP-Seq and microarray analyses identified downstream candidate genes regulated by EZH2 through the methylation of H2BK120. This is the first report to describe a novel substrate of EZH2, H2BK120, unveiling a new aspect of EZH2 functions in human carcinogenesis. Citation Format: Ryuji Hamamoto, Yusuke Nakamura. The oncogenic polycomb histone methyltransferase EZH2 methylates lysine 120 on histone H2B and competes ubiquitination in human cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5156. doi:10.1158/1538-7445.AM2014-5156

The Oncogenic Polycomb Histone Methyltransferase EZH2Methylates Lysine 120 on Histone H2B and Competes Ubiquitination

The histone methyltransferase enhancer of zeste 2 (EZH2) is known to be a polycomb protein homologous to Drosophila enhancer of zeste and catalyzes the addition of methyl groups to histone H3 at lysine 27 (H3K27). We previously reported that EZH2 was overexpressed in various types of cancer and plays a crucial role in the cell cycle regulation of cancer cells. In the present study, we demonstrated that EZH2 has the function to monomethylate lysine 120 on histone H2B (H2BK120). EZH2-dependent H2BK120 methylation in cancer cells was confirmed with an H2BK120 methylation-specific antibody. Overexpression of EZH2 significantly attenuated the ubiquitination of H2BK120, a key posttranslational modification of histones for transcriptional regulation. Concordantly, knockdown of EZH2 increased the ubiquitination level of H2BK120, suggesting that the methylation of H2BK120 by EZH2 may competitively inhibit the ubiquitination of H2BK120. Subsequent chromatin immunoprecipitation- Seq and microarray analyses identified downstream candidate genes regulated by EZH2 through the methylation of H2BK120. This is the first report to describe a novel substrate of EZH2, H2BK120, unveiling a new aspect of EZH2 functions in human carcinogenesis.

Histone H2B ubiquitin ligases RNF20 and RNF40 in androgen signaling and prostate cancer cell growth

Since data-mining from the Oncomine database revealed that expression of histone H2B K120 monoubiquitin (H2Bub1) ligase RNF20 is decreased in metastatic prostate cancer, we elucidated the effect of RNF20 and its homolog RNF40 on androgen receptor (AR)-dependent transcription and prostate cancer cell growth. Both RNF20 and RNF40 were able to functionally and physically interact with the AR and modulate its transcriptional activity in intact cells. Chromatin immunoprecipitation analyses showed that the androgen induction of FKBP51 and PSA in LNCaP prostate cancer cells is accompanied with a dynamic increase in the H2Bub1 within the transcribed regions of these loci. Interestingly, depletion of RNF20 or RNF40 strongly retarded the growth of LNCaP cells, which was however unlikely to be due to altered androgen signaling, but due to decreased expression of several cell cycle promoters. Collectively, our results suggest that RNF20 and RNF40, either via ubiquitylation of H2B or other targets, are coupled to the proliferation of prostate cancer cells.

GlcNAcylation of histone H2B facilitates its monoubiquitination

Chromatin reorganization is governed by multiple post-translational modifications of chromosomal proteins and DNA. These histone modifications are reversible, dynamic events that can regulate DNA-driven cellular processes. However, the molecular mechanisms that coordinate histone modification patterns remain largely unknown. In metazoans, reversible protein modification by O-linked N-acetylglucosamine (GlcNAc) is catalysed by two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). However, the significance of GlcNAcylation in chromatin reorganization remains elusive. Here we report that histone H2B is GlcNAcylated at residue S112 by OGT in vitro and in living cells. Histone GlcNAcylation fluctuated in response to extracellular glucose through the hexosamine biosynthesis pathway (HBP). H2B S112 GlcNAcylation promotes K120 monoubiquitination, in which the GlcNAc moiety can serve as an anchor for a histone H2B ubiquitin ligase. H2B S112 GlcNAc was localized to euchromatic areas on fly polytene chromosomes. In a genome-wide analysis, H2B S112 GlcNAcylation sites were observed widely distributed over chromosomes including transcribed gene loci, with some sites co-localizing with H2B K120 monoubiquitination. These findings suggest that H2B S112 GlcNAcylation is a histone modification that facilitates H2BK120 monoubiquitination, presumably for transcriptional activation.

MLL Fusion Protein Driven AML Is Selectively Inhibited by Targeted Disruption of the MLL-PAFc Interaction

Abstract 56Balanced chromosomal translocations of the MLL gene located on chromosome 11q23 result in the expression of a chimeric fusion proteins with enhanced transcriptional activity. The HOX genes and their co-factors, such as MEIS1 and PBX2, are critical downstream targets of MLL fusion proteins and essential for transformation. Previously we showed MLL fusion proteins are critically dependent on a direct interaction with the RNA Pol II Associated Factor complex (PAFc). PAFc is a protein complex important for the initiation, elongation and termination of transcription. It is also necessary for histone H2B K120 mono-ubiquitination through the direct recruitment of the BRE1/RAD6 E3 ubiquitin ligase complex. MLL fusion proteins make two direct contacts with the PAF1 and CTR9 subunits of the PAFc that are crucial for MLL fusion protein mediated transformation. Deletion of regions of MLL that interact with PAFc abrogates AML in mouse bone marrow transplantation assays. Here we tested the general requirement for PAFc in AML using a conditional knockout mouse model of one component of PAFc, Cdc73. These studies show that PAFc is necessary for growth of both E2A-HLF and MLL-AF9 transformed cells. Excision of Cdc73 leads to decreased expression of the MLL target genes Hoxa9 and Meis1, decreased colony formation and decreased proliferation of leukemic blasts and ultimately apoptosis. We then performed chromatin immunoprecipitation assays to assess the binding of PAFc and MLL to target loci with and without Cdc73. Excision of Cdc73 leads to a rapid decrease in association of PAFc as well as MLL fusion proteins and wild type MLL at target loci confirming that proper targeting of MLL fusion proteins requires PAFc. A decrease in H3K4me3 and H2Bub is also observed and consistent with a role of PAFc in the deposition of these epigenetic marks.We then sought to disrupt the MLL-PAFc interaction through expression of a small 40 amino acid fragment of MLL that interacts with the PAF1 subunit of PAFc. As the MLL-PAFc interaction involves interactions between MLL and both CTR9 and PAF1, it was unknown whether targeting one interaction site would be sufficient to disrupt transformation. Indeed, expression of the short fragment encompassing the pre-CxxC region of MLL acts as a dominant negative and disrupts the MLL-PAFc interaction, significantly decreasing Hox gene expression, colony formation and cell proliferation of MLL-AF9 transformed cells. Importantly, expression of the MLL fragment selectively inhibited MLL fusion mediated leukemic transformation and cell growth while the growth and proliferation of E2A-HLF cells is unaffected. Together these data show that targeting the MLL-PAFc interaction with a small MLL fragment can act as a dominant negative and selectively inhibit the growth of AML cells transformed with MLL fusion proteins. These data also suggest the MLL-PAF1 interaction surface is a promising region for therapeutic targeting. Disclosures:No relevant conflicts of interest to declare.