Polymerase Associated Factor Complex Research Articles

CTR9 drives osteochondral lineage differentiation of human mesenchymal stem cells via epigenetic regulation of BMP-2 signaling.

Cell fate determination of human mesenchymal stem/stromal cells (hMSCs) is precisely regulated by lineage-specific transcription factors and epigenetic enzymes. We found that CTR9, a key scaffold subunit of polymerase-associated factor complex (PAFc), selectively regulates hMSC differentiation to osteoblasts and chondrocytes, but not to adipocytes. An in vivo ectopic osteogenesis assay confirmed the essentiality of CTR9 in hMSC-derived bone formation. CTR9 counteracts the activity of Enhancer Of Zeste 2 (EZH2), the epigenetic enzyme that deposits H3K27me3, in hMSCs. Accordingly, CTR9 knockdown (KD) hMSCs gain H3K27me3 mark, and the osteogenic differentiation defects of CTR9 KD hMSCs can be partially rescued by treatment with EZH2 inhibitors. Transcriptome analyses identified bone morphology protein-2 (BMP-2) as a downstream effector of CTR9. BMP-2 secretion, membrane anchorage, and the BMP-SMAD pathway were impaired in CTR9 KD MSCs, and the effects were rescued by BMP-2 supplementation. This study uncovers an epigenetic mechanism engaging the CTR9-H3K27me3-BMP-2 axis to regulate the osteochondral lineage differentiation of hMSCs.

Polymerase-Associated Factor Complex (PAFc) Protects Notch-Induced Leukemia Cells from DNA Damage and Mitochondrial Stress

Polymerase-Associated Factor Complex (PAFc) Protects Notch-Induced Leukemia Cells from DNA Damage and Mitochondrial Stress

The transcriptional elongation factor CTR9 demarcates PRC2-mediated H3K27me3 domains by altering PRC2 subtype equilibrium.

CTR9 is the scaffold subunit in polymerase-associated factor complex (PAFc), a multifunctional complex employed in multiple steps of RNA Polymerase II (RNAPII)-mediated transcription. CTR9/PAFc is well known as an evolutionarily conserved elongation factor that regulates gene activation via coupling with histone modifications enzymes. However, little is known about its function to restrain repressive histone markers. Using inducible and stable CTR9 knockdown breast cancer cell lines, we discovered that the H3K27me3 levels are strictly controlled by CTR9. Quantitative profiling of histone modifications revealed a striking increase of H3K27me3 levels upon loss of CTR9. Moreover, loss of CTR9 leads to genome-wide expansion of H3K27me3, as well as increased recruitment of PRC2 on chromatin, which can be reversed by CTR9 restoration. Further, CTR9 depletion triggers a PRC2 subtype switch from the less active PRC2.2, to the more active PRC2.1 with higher methyltransferase activity. As a consequence, CTR9 depletion generates vulnerability that renders breast cancer cells hypersensitive to PRC2 inhibitors. Our findings that CTR9 demarcates PRC2-mediated H3K27me3 levels and genomic distribution provide a unique mechanism that explains the transition from transcriptionally active chromatin states to repressive chromatin states and sheds light on the biological functions of CTR9 in development and cancer.

A peptide encoded by circular form of LINC-PINT suppresses oncogenic transcriptional elongation in glioblastoma

Circular RNAs (circRNAs) are a large class of transcripts in the mammalian genome. Although the translation of circRNAs was reported, additional coding circRNAs and the functions of their translated products remain elusive. Here, we demonstrate that an endogenous circRNA generated from a long noncoding RNA encodes regulatory peptides. Through ribosome nascent-chain complex-bound RNA sequencing (RNC-seq), we discover several peptides potentially encoded by circRNAs. We identify an 87-amino-acid peptide encoded by the circular form of the long intergenic non-protein-coding RNA p53-induced transcript (LINC-PINT) that suppresses glioblastoma cell proliferation in vitro and in vivo. This peptide directly interacts with polymerase associated factor complex (PAF1c) and inhibits the transcriptional elongation of multiple oncogenes. The expression of this peptide and its corresponding circRNA are decreased in glioblastoma compared with the levels in normal tissues. Our results establish the existence of peptides encoded by circRNAs and demonstrate their potential functions in glioblastoma tumorigenesis.

The PAF complex regulation of Prmt5 facilitates the progression and maintenance of MLL fusion leukemia.

Acute myeloid leukemia (AML) is a disease associated with epigenetic dysregulation. 11q23 translocations involving the H3K4 methyltransferase MLL1 (KMT2A) generate oncogenic fusion proteins with deregulated transcriptional potential. The Polymerase Associated Factor complex (PAFc) is an epigenetic co-activator complex that makes direct contact with MLL fusion proteins and is involved in AML, however its functions are not well understood. Here, we explored the transcriptional targets regulated by the PAFc that facilitate leukemia by performing RNA-sequencing after conditional loss of the PAFc subunit Cdc73. We found Cdc73 promotes expression of an early hematopoietic progenitor gene program that prevents differentiation. Among the target genes, we confirmed the protein arginine methyltransferase Prmt5 is a direct target that is positively regulated by a transcriptional unit that includes the PAFc, MLL1, HOXA9 and STAT5 in leukemic cells. We observed reduced PRMT5-mediated H4R3me2s following excision of Cdc73 placing this histone modification downstream of the PAFc and revealing a novel mechanism between the PAFc and Prmt5. Knock down or pharmacologic inhibition of Prmt5 causes a G1 arrest and reduced proliferation resulting in extended leukemic disease latency in vivo. Overall, we demonstrate the PAFc regulates Prmt5 to facilitate leukemic progression and is a potential therapeutic target for AMLs.

Super elongation complex promotes early HIV transcription and its function is modulated by P-TEFb

ABSTRACTEarly work on the control of transcription of the human immunodeficiency virus (HIV) laid the foundation for our current knowledge of how RNA Polymerase II is released from promoter-proximal pausing sites and transcription elongation is enhanced. The viral Tat activator recruits Positive Transcription Elongation Factor b (P-TEFb) and Super Elongation Complex (SEC) that jointly drive transcription elongation. While substantial progress in understanding the role of SEC in HIV gene transcription elongation has been obtained, defining of the mechanisms that govern SEC functions is still limited, and the role of SEC in controlling HIV transcription in the absence of Tat is less clear.Here we revisit the contribution of SEC in early steps of HIV gene transcription. In the absence of Tat, the AF4/FMR2 Family member 4 (AFF4) of SEC efficiently activates HIV transcription, while gene activation by its homolog AFF1 is substantially lower. Differential recruitment to the HIV promoter and association with Human Polymerase-Associated Factor complex (PAFc) play key role in this functional distinction between AFF4 and AFF1. Moreover, while depletion of cyclin T1 expression has subtle effects on HIV gene transcription in the absence of Tat, knockout (KO) of AFF1, AFF4, or both proteins slightly repress this early step of viral transcription. Upon Tat expression, HIV transcription reaches optimal levels despite KO of AFF1 or AFF4 expression. However, double AFF1/AFF4 KO completely diminishes Tat trans-activation. Significantly, our results show that P-TEFb phosphorylates AFF4 and modulates SEC assembly, AFF1/4 dimerization and recruitment to the viral promoter.We conclude that SEC promotes both early steps of HIV transcription in the absence of Tat, as well as elongation of transcription, when Tat is expressed. Significantly, SEC functions are modulated by P-TEFb.

The PAF Complex Regulation of Prmt5 Facilitates Leukemic Progression

Acute myeloid leukemias have been linked with dysregulated epigenetic landscapes sometimes attributed to altered functions of epigenetic regulators. The Polymerase-Associated Factor complex (PAFc) is an epigenetic regulator involved in transcriptional initiation, elongation and termination and directly interacts with the CTD of RNA Pol II. The complex is comprised of 6 subunits in human cells, Paf1, Cdc73, Ctr9, Leo1, Rtf1 and Ski8. Many of these subunits have key roles in a variety of cancers including acute myeloid leukemia (AML). We have previously shown the relevance of the PAFc in MLL-rearranged leukemias where its interaction with MLL fusion-proteins is required for leukemic progression in vitro and in vivo (Muntean et al. 2013 Blood, Muntean et al. 2010 Cancer Cell). However, little is known about the gene programs controlled by the PAFc and how these contribute to leukemogenesis.Here we identify Prmt5, an arginine methyltransferase, as a direct downstream target gene of the PAFc. Prmt5 is upregulated in variety of cancers and has been linked to cell cycle progression and activation of known oncoproteins. In addition, Prmt5 has been implicated in AML and is essential for normal hematopoiesis where loss of Prmt5 induces bone marrow aplasia due to impaired cytokine signaling (Tarighat et al. 2015 Leukemia, Liu et al. 2015 J Clin Invest). Our work establishes a major role for the PAFc in regulating Prmt5 expression in AML. We observe that excision of the Cdc73 subunit of the PAFc results in reduced proliferation, the induction of differentiation, cell cycle arrest, and a mild increase in apoptosis. Several key epigenetic marks are reduced globally upon loss of Cdc73 including H4R3me2s, a modification catalyzed by Prmt5. RNA sequencing and bioinformatics analysis using GSEA, revealed that loss of Cdc73 led to increased expression of a gene program associated with hematopoietic differentiation, in agreement with our cellular characterization. In addition, the downregulation of a methyltransferase gene program was detected upon Cdc73 excision. Included in this signature were several members of the Prmt family. Analysis of changes in expression following loss of Cdc73 and functional relevance in MLL-AF9 leukemic cells led us to Prmt5 as a gene critically important in AML cells and modulated by the PAFc. To interrogate the function of Prmt5 in AML cells, we performed shRNA knockdown experiments which resulted in reduced proliferation, reduced cell fitness, G1 cell cycle arrest and global reduction H4R3me2s. ChIP experiments revealed that the PAFc localizes to the Prmt5 locus in mouse and human derived leukemic cells. Further, preliminary data suggests the MLL-AF9 fusion protein also localizes to the Prmt5 locus and may enhance its transcriptional output. The enzymatic activity of Prmt5 is necessary for AML cell growth as wild type PRMT5 can rescue proliferation of Prmt5 knock-down cells while a catalytic dead mutant cannot. Furthermore, we have observed that knockdown of Prmt5 increases the disease latency of Hoxa9/Meis1 induced leukemia in vivo. Utilizing a commercially available inhibitor for Prmt5, EPZ015666 (Chan-Pembre et al. 2015 Nat Chem Bio), we show pharmacologic inhibition of PRMT5 reduces the growth of a spectrum of human leukemic cell lines, suggesting PRMT5 is important for multiple subtypes of AML. Overall, our findings elucidate the PAFc as a regulator of Prmt5 expression that is necessary for the maintenance of AML. DisclosuresNo relevant conflicts of interest to declare.

Cln 3-requiring 9 is a negative regulator of Th17 pathway-driven inflammation in anti-glomerular basement membrane glomerulonephritis.

T helper 17 (Th17) lymphocytes promote renal inflammation in anti-glomerular basement membrane glomerulonephritis (anti-GBM GN), and signal transducer and activator of transcription 3 (STAT3) mediates activation of Th17 lymphocytes by IL-6 and transforming growth factor-β (TGF-β). Cln 3-requiring 9 (Ctr9), a subunit of RNA polymerase-associated factor complex (PAFc), regulates the transcription of IL-6/STAT3-dependent genes. Here, we investigated the role of Ctr9 in regulating Th17-driven inflammation in anti-GBM GN. In mice, STAT3β or IL-17 knockout ameliorated anti-GBM autoantibody-induced renal injury. This phenomenon was associated with decreases in retinoic acid receptor-related orphan receptor γt (RORγt), IL-17, phosphorylated STAT3, and proinflammatory cytokines. Compared with wild-type mice, Ctr9 increased in both STAT3β(-/-) and IL-17(-/-) mice injected with anti-GBM IgG, showing a negative correlation with Th17-related transcripts. Small interfering RNA (siRNA)-mediated knockdown of Ctr9 in intrarenal lymphocytes further upregulated Th17-related transcripts, consistent with repression of Th17 differentiation by Ctr9. Interestingly, Ctr9 was also expressed in human and mouse mesangial cells and downregulated in response to anti-GBM IgG or to TGF-β plus IL-17. Ctr9 in mesangial cells was even more repressed in the presence of both anti-GBM IgG and Th17-activating cytokines. Consistent with these findings, renal biopsies obtained from patients with anti-GBM GN showed consistent downregulation of Ctr9 and upregulation of phosphorylated STAT3 and IL-17 in the glomerulus. We conclude that Ctr9 is a negative regulator of Th17 differentiation in anti-GBM GN and repressed by anti-GBM IgG and IL-17 in mesangial cells.

Quantitative Analysis of Dynamic Protein Interactions during Transcription Reveals a Role for Casein Kinase II in Polymerase-associated Factor (PAF) Complex Phosphorylation and Regulation of Histone H2B Monoubiquitylation

Using affinity purification MS approaches, we have identified a novel role for casein kinase II (CKII) in the modification of the polymerase associated factor complex (PAF-C). Our data indicate that the facilitates chromatin transcription complex (FACT) interacts with CKII and may facilitate PAF complex phosphorylation. Posttranslational modification analysis of affinity-isolated PAF-C shows extensive CKII phosphorylation of all five subunits of PAF-C, although CKII subunits were not detected as interacting partners. Consistent with this, recombinant CKII or FACT-associated CKII isolated from cells can phosphorylate PAF-C in vitro, whereas no intrinsic kinase activity was detected in PAF-C samples. Significantly, PAF-C purifications combined with stable isotope labeling in cells (SILAC) quantitation for PAF-C phosphorylation from wild-type and CKII temperature-sensitive strains (cka1Δ cka2-8) showed that PAF-C phosphorylation at consensus CKII sites is significantly reduced in cka1Δ cka2-8 strains. Consistent with a role of CKII in FACT and PAF-C function, we show that decreased CKII function in vivo results in decreased levels of histone H2B lysine 123 monoubiquitylation, a modification dependent on FACT and PAF-C. Taken together, our results define a coordinated role of CKII and FACT in the regulation of RNA polymerase II transcription through chromatin via phosphorylation of PAF-C.

The PAF Complex and Prf1/Rtf1 Delineate Distinct Cdk9-Dependent Pathways Regulating Transcription Elongation in Fission Yeast

Cyclin-dependent kinase 9 (Cdk9) promotes elongation by RNA polymerase II (RNAPII), mRNA processing, and co-transcriptional histone modification. Cdk9 phosphorylates multiple targets, including the conserved RNAPII elongation factor Spt5 and RNAPII itself, but how these different modifications mediate Cdk9 functions is not known. Here we describe two Cdk9-dependent pathways in the fission yeast Schizosaccharomyces pombe that involve distinct targets and elicit distinct biological outcomes. Phosphorylation of Spt5 by Cdk9 creates a direct binding site for Prf1/Rtf1, a transcription regulator with functional and physical links to the Polymerase Associated Factor (PAF) complex. PAF association with chromatin is also dependent on Cdk9 but involves alternate phosphoacceptor targets. Prf1 and PAF are biochemically separate in cell extracts, and genetic analyses show that Prf1 and PAF are functionally distinct and exert opposing effects on the RNAPII elongation complex. We propose that this opposition constitutes a Cdk9 auto-regulatory mechanism, such that a positive effect on elongation, driven by the PAF pathway, is kept in check by a negative effect of Prf1/Rtf1 and downstream mono-ubiquitylation of histone H2B. Thus, optimal RNAPII elongation may require balanced action of functionally distinct Cdk9 pathways.

MLL fusion protein–driven AML is selectively inhibited by targeted disruption of the MLL-PAFc interaction

AbstractMLL rearrangements are common in leukemia and considered an adverse risk factor. Through interactions with the polymerase-associated factor complex (PAFc), mixed lineage leukemia (MLL) fusion proteins activate genes critical for blocking differentiation, such as HOXA9. Here we investigate whether the MLL-PAFc interaction can be exploited therapeutically using both genetic and biochemical approaches. We tested the genetic requirement of the PAFc in acute myeloid leukemia (AML) using a conditional allele of the PAFc subunit, Cdc73. We show that the PAFc is indiscriminately necessary for the proliferation of AML cells through the epigenetic regulation of proleukemogenic target genes, such as MEIS1 and Bcl2. To investigate the therapeutic potential of targeting the MLL-PAFc interaction, we engineered a dominant negative fragment of MLL capable of binding to the PAFc. Disruption of the MLL-PAFc interaction selectively inhibits the proliferation of MLL leukemic cells without affecting cells transformed by an unrelated E2A-HLF fusion protein. Using in vivo hematopoietic reconstitution assays, we demonstrate that disruption of the MLL-PAFc does not alter normal hematopoietic stem cell function. Together, our data show a selective growth inhibition of MLL-associated leukemic cells and tolerance of normal hematopoiesis to disruption of the MLL-PAFc interaction establishing the MLL-PAFc interaction as an attractive therapeutic target.

Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia.

Recurrent chromosomal translocations involving the mixed lineage leukaemia (MLL) gene initiate aggressive forms of leukaemia, which are often refractory to conventional therapies. Many MLL-fusion partners are members of the super elongation complex (SEC), a critical regulator of transcriptional elongation, suggesting that aberrant control of this process has an important role in leukaemia induction. Here we use a global proteomic strategy to demonstrate that MLL fusions, as part of SEC and the polymerase-associated factor complex (PAFc), are associated with the BET family of acetyl-lysine recognizing, chromatin 'adaptor' proteins. These data provided the basis for therapeutic intervention in MLL-fusion leukaemia, via the displacement of the BET family of proteins from chromatin. We show that a novel small molecule inhibitor of the BET family, GSK1210151A (I-BET151), has profound efficacy against human and murine MLL-fusion leukaemic cell lines, through the induction of early cell cycle arrest and apoptosis. I-BET151 treatment in two human leukaemia cell lines with different MLL fusions alters the expression of a common set of genes whose function may account for these phenotypic changes. The mode of action of I-BET151 is, at least in part, due to the inhibition of transcription at key genes (BCL2, C-MYC and CDK6) through the displacement of BRD3/4, PAFc and SEC components from chromatin. In vivo studies indicate that I-BET151 has significant therapeutic value, providing survival benefit in two distinct mouse models of murine MLL-AF9 and human MLL-AF4 leukaemia. Finally, the efficacy of I-BET151 against human leukaemia stem cells is demonstrated, providing further evidence of its potent therapeutic potential. These findings establish the displacement of BET proteins from chromatin as a promising epigenetic therapy for these aggressive leukaemias.

Human Polymerase-Associated Factor complex (PAFc) connects the Super Elongation Complex (SEC) to RNA polymerase II on chromatin

The Super Elongation Complex (SEC), containing transcription elongation activators/coactivators P-TEFb, ELL2, AFF4/1, ENL, and AF9, is recruited by HIV-1 Tat and mixed lineage leukemia (MLL) proteins to activate the expression of HIV-1 and MLL-target genes, respectively. In the absence of Tat and MLL, however, it is unclear how SEC is targeted to RNA polymerase (Pol) II to stimulate elongation in general. Furthermore, although ENL and AF9 can bind the H3K79 methyltransferase Dot1L, it is unclear whether these bindings are required for SEC-mediated transcription. Here, we show that the homologous ENL and AF9 exist in separate SECs with similar but nonidentical functions. ENL/AF9 contacts the scaffolding protein AFF4 that uses separate domains to recruit different subunits into SEC. ENL/AF9 also exists outside SEC when bound to Dot1L, which is found to inhibit SEC function. The YEATS domain of ENL/AF9 targets SEC to Pol II on chromatin through contacting the human Polymerase-Associated Factor complex (PAFc) complex. This finding explains the YEATS domain's dispensability for leukemogenesis when ENL/AF9 is translocated to MLL, whose interactions with PAFc and DNA likely substitute for the PAFc/chromatin-targeting function of the YEATS domain.

CBX8, a Polycomb-Group Protein, Is Essential for MLL-AF9-Induced Leukemogenesis

AbstractAbstract 4174Trithorax and Polycomb-group (Trx-G and Pc-G) proteins are antagonistic regulators of homeobox-containing (Hox) gene expression that play a major role in regulation of hematopoiesis and leukemogenesis. Mixed lineage leukemia (MLL), a mammalian Trx-G protein, is a histone methyltransferase crucial for embryonic development and hematopoiesis that is commonly altered by translocation in acute leukemia. Recent evidence suggests that transformation by MLL fusion proteins is dependent on multiple interaction complexes, including the polymerase associated factor complex (PAFc) and the elongation activating protein complex (EAPc) or a closely related AF4 family/ENL family/P-TEFb complex (AEPc). CBX8 is a human PcG protein, functioning as a transcription repressor in the polycomb repressive complex 1 (PRC1). Previous studies have shown that CBX8 also interacts with the EAPc components AF9 and ENL; however, its role in leukemogenesis is unknown. To elucidate the significance of this interaction between these two proteins thought to have antagonistic function, we generated a large series of point mutations in AF9 and identified two amino acids that are essential for CBX8 interaction but preserve the interaction with other EAP components. Mutation of the two sites reduced the transcriptional activation of the MLL-AF9 target promoters by nearly 50% and completely inhibits the ability of MLL-AF9 to immortalize bone marrow (BM) as assessed by methylcellulose replating assays. This finding suggests that CBX8 interaction is essential for MLL-AF9-induced leukemogenesis. Several lines of evidence further support this finding. First, CBX8 knockdown by siRNAs decreased MLL-AF9-induced transcriptional activation by approximately 50%. Second, the ability of MLL-AF9 to transform primary BM was markedly reduced by retroviral shCbx8 transduction. Notably, this inhibitory effect is specific for MLL-AF9 because the BM transformation ability of E2A-HLF was unaffected by Cbx8 suppression. Third, Cbx8 suppression by shCbx8 in MLL-AF9 and MLL-ENL, but not E2A-HLF transformed AML cell lines, significantly inhibited the expression of MLL-dependent target genes, as well as cell growth and colony forming ability. Fourth, inducing CBX8 knockdown in human leukemia cell lines expressing MLL-AF9 led to a marked decrease in the localization of basic transcription machinery at the Hoxa9 locus and a corresponding reduction in Hoxa9 transcription. Importantly, the observed effects of CBX8 on MLL-rearranged leukemia cells are PRC1-independent: no effects on MLL target gene expression, cell growth, or BM transformation ability were observed by suppressing other core components of PRC1. Taken together, our results indicate that CBX8, independent of its transcription repression role in PRC1, interacts with and synergizes with MLL fusion proteins to promote leukemogenesis. Defining the interaction sites between AF9/ENL and CBX8 and the dependence of other AML subtypes and normal hematopoiesis on CBX8 will be important for the further development of agents that target this mechanism in MLL-rearranged and potentially other AML subtypes. Disclosures:No relevant conflicts of interest to declare.

PAFc, a Key Player in MLL-rearranged Leukemogenesis

Recent studies identified an interaction between the Polymerase Associated Factor complex (PAFc) and Mixed Lineage Leukemia (MLL), including MLL-rearranged oncoproteins. This interaction is critical for MLL transcriptional activity and MLL-rearranged leukemogenesis. Here, we discuss the potential molecular role of the PAFc in transcriptional dysregulation of MLL target genes and the interplay between PAFc and MLL-rearranged oncoproteins in leukemogenesis.

The PAF Complex Synergizes with MLL Fusion Proteins at HOX Loci to Promote Leukemogenesis

MLL is involved in chromosomal rearrangements that generate fusion proteins with deregulated transcriptional activity. The mechanisms of MLL fusion protein-mediated transcriptional activation are poorly understood. Here we show MLL interacts directly with the polymerase associated factor complex (PAFc) through sequences flanking the CxxC domain. PAFc interacts with RNA polymerase II and stimulates posttranslational histone modifications. PAFc augments MLL and MLL-AF9 mediated transcriptional activation of Hoxa9. Conversely, knockdown of PAFc disrupts MLL fusion protein-mediated transcriptional activation and MLL recruitment to target loci. PAFc gene expression is downregulated during hematopoiesis and likely serves to regulate MLL function. Deletions of MLL that abolish interactions with PAFc also eliminate MLL-AF9 mediated immortalization indicating an essential function for this interaction in leukemogenesis.

The PAF Complex Synergizes with MLL Fusion Proteins at .Hox Loci to Promote Leukemogenesis.

Abstract 1277Poster Board I-299Mixed lineage leukemia (MLL) is a histone H3 lysine 4 methyltransferase that is required to maintain a normal hematopoietic stem cell compartment. MLL functions to maintain expression of HOX genes as well as the HOX co-factor MEIS1, which play significant roles in regulating hematopoiesis. MLL is involved in chromosomal translocations with up to sixty different partners in both acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). HOXA9 and MEIS1, are directly regulated by MLL fusion proteins and are crucial for MLL fusion protein mediated transformation. The deregulated expression of target genes in AML is dependent on specific protein-protein interactions and functional domains of MLL. For example, the tumor suppressor Menin bridges LEDGF to the extreme N-terminus of MLL and both of these interactions are necessary for transformation. Furthermore, a DNA methyltransferase homology region (CxxC domain) of MLL is essential for binding to non-methylated CpG islands and MLL-fusion protein oncogenesis. We have found that sequences downstream of the CxxC domain, termed the RD2 region, that interact with the Polymerase Associated Factor (PAF) complex are also required for MLL fusion protein mediated transformation. The PAF complex interacts with RNA polymerase II and is required for H2B mono-ubiquitination and subsequent histone H3K4 and H3K79 methylation. Together the PAF complex has been shown to be involved in transcriptional initiation, elongation and termination. Interaction of MLL with the PAF complex is mediated through direct contacts with two subunits: Ctr9 and PAF1. The PAF complex synergizes with MLL-AF9 to augment transcriptional activation of the Hoxa9 promoter. Furthermore, MLL fusion proteins recruit high levels of the PAF complex to the Hoxa9 promoter. Importantly, deletions of the MLL RD2 region that abolish interactions with the PAF complex eliminate MLL-AF9 mediated transformation of mouse bone marrow cells. Transcription of PAF components is dramatically downregulated during differentiation of hematopoietic cells, consistent with recent data showing a requirement for the PAF complex to maintain an embryonic stem cell phenotype. Knock down and transplantation experiments are underway to further define how the PAF complex regulates normal MLL function and cooperates with MLL fusion proteins to promote leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.

Control of transcriptional elongation and cotranscriptional histone modification by the yeast BUR kinase substrate Spt5

Elongation by RNA polymerase II (RNAPII) is a finely regulated process in which many elongation factors contribute to gene regulation. Among these factors are the polymerase-associated factor (PAF) complex, which associates with RNAPII, and several cyclin-dependent kinases, including positive transcription elongation factor b (P-TEFb) in humans and BUR kinase (Bur1-Bur2) and C-terminal domain (CTD) kinase 1 (CTDK1) in Saccharomyces cerevisiae. An important target of P-TEFb and CTDK1, but not BUR kinase, is the CTD of the Rpb1 subunit of RNAPII. Although the essential BUR kinase phosphorylates Rad6, which is required for histone H2B ubiquitination on K123, Rad6 is not essential, leaving a critical substrate(s) of BUR kinase unidentified. Here we show that BUR kinase is important for the phosphorylation in vivo of Spt5, a subunit of the essential yeast RNAPII elongation factor Spt4/Spt5, whose human orthologue is DRB sensitivity-inducing factor. BUR kinase can also phosphorylate the C-terminal region (CTR) of Spt5 in vitro. Like BUR kinase, the Spt5 CTR is important for promoting elongation by RNAPII and recruiting the PAF complex to transcribed regions. Also like BUR kinase and the PAF complex, the Spt5 CTR is important for histone H2B K123 monoubiquitination and histone H3 K4 and K36 trimethylation during transcription elongation. Our results suggest that the Spt5 CTR, which contains 15 repeats of a hexapeptide whose consensus sequence is S[T/A]WGG[A/Q], is a substrate of BUR kinase and a platform for the association of proteins that promote both transcription elongation and histone modification in transcribed regions.