New twists of a TAIL: novel insights into the histone binding properties of a highly conserved PHD finger cluster within the MLR family of H3K4 mono-methyltransferases.

Enhancer activation by the MLR family of H3K4 mono-methyltransferases requires proper recognition of histones for the deposition of the mono-methyl mark. MLR proteins contain two clusters of PHD zinc finger domains implicated in chromatin regulation. The second cluster is the most highly conserved, preserved as an ancient three finger functional unit throughout evolution. Studies of the isolated 3rd PHD finger within this cluster suggested specificity for the H4 [aa16-20] tail region. We determined the histone binding properties of the full three PHD finger cluster b module (PHDb) from the Drosophila Cmi protein which revealed unexpected recognition of an extended region of H3. Importantly, the zinc finger spacer separating the first two PHDb fingers from the third is critical for proper alignment and coordination among fingers for maximal histone engagement. Human homologs, MLL3 and MLL4, also show conservation of H3 binding, expanding current views of histone recognition for this class of proteins. We further implicate chromatin remodeling by the SWI/SNF complex as a possible mechanism for the accessibility of PHDb to globular regions of histone H3 beyond the tail region. Our results suggest a two-tail histone recognition mechanism by the conserved PHDb domain involving a flexible hinge to promote interdomain coordination.

RNA binding induces an allosteric switch in Cyp33 to repress MLL1-mediated transcription.

Mixed-lineage leukemia 1 (MLL1) is a transcription activator of the HOX family, which binds to specific epigenetic marks on histone H3 through its third plant homeodomain (PHD3) domain. Through an unknown mechanism, MLL1 activity is repressed by cyclophilin 33 (Cyp33), which binds to MLL1 PHD3. We determined solution structures of Cyp33 RNA recognition motif (RRM) free, bound to RNA, to MLL1 PHD3, and to both MLL1 and the histone H3 lysine N6-trimethylated. We found that a conserved α helix, amino-terminal to the RRM domain, adopts three different positions facilitating a cascade of binding events. These conformational changes are triggered by Cyp33 RNA binding and ultimately lead to MLL1 release from the histone mark. Together, our mechanistic findings rationalize how Cyp33 binding to MLL1 can switch chromatin to a transcriptional repressive state triggered by RNA binding as a negative feedback loop.

Multiple clonal MLL fusions in a patient receiving CHOP-based chemotherapy.

MLL rearrangements were analysed in the blood of a patient receiving chemotherapy for diffuse large B-cell lymphoma using inverse polymerase chain reaction targeting exon 12, parallel sequencing and a custom algorithm design. Of thirteen MLL rearrangements detected, five were capable of generating MLL fusion genes, including MLL-MLLT3, the most common fusion in acute myeloid leukaemia (AML). Other fusions, all previously clinically unobserved, included MLL-NKD1, a fusion to the negative regulator of Wnt/ß-catenin signaling, a pathway linked to leukaemic cell proliferation. The majority of the fusions exhibited clonal persistence from before treatment until 6 months post-chemotherapy, suggesting the fusions may confer a survival advantage to the mutant clone. MLL breakpoints were partly clustered at a specific location, indicating commonality in the process of their formation. Further, the same MLL breakpoint location exhibited a 50-100-fold increase in C to T transitions, consistent with attack by activation-induced cytidine deaminase (AICDA). As is also observed in AML and acute lymphoblastic leukaemia, in this single patient setting, MLL is capable of interacting with multiple fusion partners. This finding defines a discrete site of MLL susceptibility to fragmentation, linked to possible deregulation of AICDA function.

Histone recognition and nuclear receptor co-activator functions of Drosophila cara mitad, a homolog of the N-terminal portion of mammalian MLL2 and MLL3.

MLL2 and MLL3 histone lysine methyltransferases are conserved components of COMPASS-like co-activator complexes. In vertebrates, the paralogous MLL2 and MLL3 contain multiple domains required for epigenetic reading and writing of the histone code involved in hormone-stimulated gene programming, including receptor-binding motifs, SET methyltransferase, HMG and PHD domains. The genes encoding MLL2 and MLL3 arose from a common ancestor. Phylogenetic analyses reveal that the ancestral gene underwent a fission event in some Brachycera dipterans, including Drosophila species, creating two independent genes corresponding to the N- and C-terminal portions. In Drosophila, the C-terminal SET domain is encoded by trithorax-related (trr), which is required for hormone-dependent gene activation. We identified the cara mitad (cmi) gene, which encodes the previously undiscovered N-terminal region consisting of PHD and HMG domains and receptor-binding motifs. The cmi gene is essential and its functions are dosage sensitive. CMI associates with TRR, as well as the EcR-USP receptor, and is required for hormone-dependent transcription. Unexpectedly, although the CMI and MLL2 PHDf3 domains could bind histone H3, neither showed preference for trimethylated lysine 4. Genetic tests reveal that cmi is required for proper global trimethylation of H3K4 and that hormone-stimulated transcription requires chromatin binding by CMI, methylation of H3K4 by TRR and demethylation of H3K27 by the demethylase UTX. The evolutionary split of MLL2 into two distinct genes in Drosophila provides important insight into distinct epigenetic functions of conserved readers and writers of the histone code.

The PHD3 domain of MLL acts as a CYP33-regulated switch between MLL-mediated activation and repression .

The mixed lineage leukemia (MLL) gene plays a critical role in epigenetic regulation of gene expression and is a frequent target of chromosomal translocations leading to leukemia. MLL plant homeodomain 3 (PHD3) is lost in all MLL translocation products, and reinsertion of PHD3 into MLL fusion proteins abrogates their transforming activity. PHD3 has been shown to interact with the RNA-recognition motif (RRM) domain of human nuclear Cyclophilin33 (CYP33). Here, we show that CYP33 mediates downregulation of the expression of MLL target genes HOXC8, HOXA9, CDKN1B, and C-MYC, in a proline isomerase-dependent manner. This downregulation correlates with the reduction of trimethylated lysine 4 of histone H3 (H3K4me3) and histone H3 acetylation. We have structurally characterized both the PHD3 and CYP33 RRM domains and analyzed their binding to one another. The PHD3 domain binds H3K4me3 (preferentially) and the CYP33 RRM domain at distinct sites. Our binding data show that binding of H3K4me3 to PHD3 and binding of the CYP33 RRM domain to PHD3 are mutually inhibitory, implying that PHD3 is a molecular switch for the transition between activation and repression of target genes. To explore the possible mechanism of CYP33/PHD3-mediated repression, we have analyzed the CYP33 proline isomerase activity on various H3 and H4 peptides and shown selectivity for two sites in H3. Our results provide a possible mechanism for the MLL PHD3 domain to act as a switch between activation and repression.

CBP-mediated acetylation of histone H3 lysine 27 antagonizes Drosophila Polycomb silencing.

Trimethylation of histone H3 lysine 27 (H3K27me3) by Polycomb repressive complex 2 (PRC2) is essential for transcriptional silencing of Polycomb target genes, whereas acetylation of H3K27 (H3K27ac) has recently been shown to be associated with many active mammalian genes. The Trithorax protein (TRX), which associates with the histone acetyltransferase CBP, is required for maintenance of transcriptionally active states and antagonizes Polycomb silencing, although the mechanism underlying this antagonism is unknown. Here we show that H3K27 is specifically acetylated by Drosophila CBP and its deacetylation involves RPD3. H3K27ac is present at high levels in early embryos and declines after 4 hours as H3K27me3 increases. Knockdown of E(Z) decreases H3K27me3 and increases H3K27ac in bulk histones and at the promoter of the repressed Polycomb target gene abd-A, suggesting that these indeed constitute alternative modifications at some H3K27 sites. Moderate overexpression of CBP in vivo causes a global increase in H3K27ac and a decrease in H3K27me3, and strongly enhances Polycomb mutant phenotypes. We also show that TRX is required for H3K27 acetylation. TRX overexpression also causes an increase in H3K27ac and a concomitant decrease in H3K27me3 and leads to defects in Polycomb silencing. Chromatin immunoprecipitation coupled with DNA microarray (ChIP-chip) analysis reveals that H3K27ac and H3K27me3 are mutually exclusive and that H3K27ac and H3K4me3 signals coincide at most sites. We propose that TRX-dependent acetylation of H3K27 by CBP prevents H3K27me3 at Polycomb target genes and constitutes a key part of the molecular mechanism by which TRX antagonizes or prevents Polycomb silencing.

Rearrangements of the MLL gene are influenced by DNA secondary structure, potentially mediated by topoisomerase II binding.

The location of MLL translocation breakpoints within therapy-related acute myeloid leukemia linked to drugs targeting Topoisomerase II and infant acute leukemia (IAL) are biased toward the intron 11-exon 12 region of MLL, although lacking a comprehensive explanation. To address this, blood samples were taken from breast cancer and lymphoma patients receiving Topoisomerase II inhibitor therapy. Inverse PCR analysis was used to interrogate the exon 12 region of MLL for rearrangements. Eleven of 19 observed translocations showed breakpoint junctions restricted to a single 5 bp location within exon 12. A similarly restricted distribution (11/20 breakpoint junctions) was observed in TK6 cells exposed to either estrogen (linked to IAL) or anti-CD95 antibody. The translocation hotspot was at the 5' edge of a 10-bp tract matched with a perfect palindrome, 101 bp distant. A high stringency Topoisomerase II consensus sequence binding site was noted at the geometric midpoint of the palindromes. Ligation-mediated PCR to screen TK6 cells exposed to anti-CD95 antibody showed 14/37 (38%) of DNA breaks adjacent to the 5' palindrome and 10/37 (27%) at the 3' partner. We propose a model whereby Topoisomerase II facilitates the organization of nuclease-sensitive secondary structures, stabilized by palindrome association, which are prone to rearrangement.

Loss of MLL PHD finger 3 is necessary for MLL-ENL-induced hematopoietic stem cell immortalization.

Reciprocal chromosomal translocations at the MLL gene locus result in expression of novel fusion proteins, such as MLL-ENL, associated with leukemia. The three PHD finger cassette, one of the highly conserved domains in MLL, is absent in all fusion proteins. This domain has been shown to interact with Cyp33, a cyclophilin which enhances the recruitment of histone deacetylases (HDAC) to the MLL repression domain and mediates HOX gene repression. Insertion of the third PHD finger of MLL into MLL-ENL allows the recruitment of Cyp33 and, subsequently, HDAC1 to the fusion protein. Furthermore, expression of the fusion protein with the PHD finger insertion mediates the down-regulation of the HOXC8 gene expression in a Cyp33-dependent manner. Finally, the addition of the PHD finger domain or the third PHD finger alone into MLL-ENL blocks the hematopoietic stem cell immortalization potential of the fusion protein in serial plating colony assays. Insertion of only the first and second PHD fingers has no such effect. These data support the hypothesis that the binding of Cyp33 to the MLL third PHD finger switches the MLL function from transactivation to repression. In the immortalizing MLL fusion protein, the loss of the PHD fingers, in combination with the gain of the activation domain of ENL or of other partner proteins, makes the fusion protein a constitutive transactivator. This leads to constitutive overexpression of MLL target genes that block stem cell commitment and promote stem cell renewal, probably the first step in MLL-related leukemogenesis.

MLL protects CpG clusters from methylation within the Hoxa9 gene, maintaining transcript expression.

Homeobox (HOX) genes play a definitive role in determination of cell fate during embryogenesis and hematopoiesis. MLL-related leukemia is coincident with increased expression of a subset of HOX genes, including HOXA9. MLL functions to maintain, rather than initiate, expression of its target genes. However, the mechanism of MLL maintenance of target gene expression is not understood. Here, we demonstrate that Mll binds to specific clusters of CpG residues within the Hoxa9 locus and regulates expression of multiple transcripts. The presence of Mll at these clusters provides protection from DNA methylation. shRNA knock-down of Mll reverses the methylation protection status at the previously protected CpG clusters; methylation at these CpG residues is similar to that observed in Mll null cells. Furthermore, reconstituting MLL expression in Mll null cells can reverse DNA methylation of the same CpG residues, demonstrating a dominant effect of MLL in protecting this specific region from DNA methylation. Intriguingly, an oncogenic MLL-AF4 fusion can also reverse DNA methylation, but only for a subset of these CpGs. This method of transcriptional regulation suggests a mechanism that explains the role of Mll in transcriptional maintenance, but it may extend to other CpG DNA binding proteins. Protection from methylation may be an important mechanism of epigenetic inheritance by regulating the function of both de novo and maintenance DNA methyltransferases.

The MLL fusion gene, MLL-AF4, regulates cyclin-dependent kinase inhibitor CDKN1B (p27kip1) expression.

MLL, involved in many chromosomal translocations associated with acute myeloid and lymphoid leukemia, has >50 known partner genes with which it is able to form in-frame fusions. Characterizing important downstream target genes of MLL and of MLL fusion proteins may provide rational therapeutic strategies for the treatment of MLL-associated leukemia. We explored downstream target genes of the most prevalent MLL fusion protein, MLL-AF4. To this end, we developed inducible MLL-AF4 fusion cell lines in different backgrounds. Overexpression of MLL-AF4 does not lead to increased proliferation in either cell line, but rather, cell growth was slowed compared with similar cell lines inducibly expressing truncated MLL. We found that in the MLL-AF4-induced cell lines, the expression of the cyclin-dependent kinase inhibitor gene CDKN1B was dramatically changed at both the RNA and protein (p27kip1) levels. In contrast, the expression levels of CDKN1A (p21) and CDKN2A (p16) were unchanged. To explore whether CDKN1B might be a direct target of MLL and of MLL-AF4, we used chromatin immunoprecipitation (ChIP) assays and luciferase reporter gene assays. MLL-AF4 binds to the CDKN1B promoter in vivo and regulates CDKN1B promoter activity. Further, we confirmed CDKN1B promoter binding by ChIP in MLL-AF4 as well as in MLL-AF9 leukemia cell lines. Our results suggest that CDKN1B is a downstream target of MLL and of MLL-AF4, and that, depending on the background cell type, MLL-AF4 inhibits or activates CDKN1B expression. This finding may have implications in terms of leukemia stem cell resistance to chemotherapy in MLL-AF4 leukemias.

Surviving apoptosis: a possible mechanism of benzene-induced leukemia.

The pathological consequences resulting from deregulation of the apoptotic program include cancer (too little apoptosis) or diseases of cell deprivation, such as Alzheimer's (too much apoptosis). We have identified an additional pathology whereby cells reaching the earliest stage of chromatin cleavage have the potential to suppress apoptotic execution and survive. One specific cleavage event associated with this process is restricted to a location within the mixed lineage leukemia (MLL) gene at 11q23. The site of cleavage is consistent with the location where large, approximately 50 kbp loops of supercoiled DNA are attached to the nuclear matrix. Cells modified by this process generate MLL translocations, as shown by inverse PCR, that survive for days to weeks but which have no known relationship with clinical disease. Using a specific approach, cells stimulated by anti-CD95 antibody, a potent stimulator of the apoptotic program, facilitated creation of the MLL-AF9 fusion gene. Further, this rearrangement, which is commonly observed in patients with AML linked to exposure to cytotoxic agents, was efficiently transcribed in cells that were able to undergo cell division. These data are discussed in the context of benzene and benzene metabolite toxicity that impacts the process of apoptosis and is known to lead to leukemic disease.

MLL repression domain interacts with histone deacetylases, the polycomb group proteins HPC2 and BMI-1, and the corepressor C-terminal-binding protein.

The MLL (mixed-lineage leukemia) gene is involved in many chromosomal translocations associated with acute myeloid and lymphoid leukemia. We previously identified a transcriptional repression domain in MLL, which contains a region with homology to DNA methyltransferase. In chromosomal translocations, the MLL repression domain is retained in the leukemogenic fusion protein and is required for transforming activity of MLL fusion proteins. We explored the mechanism of action of the MLL repression domain. Histone deacetylase 1 interacts with the MLL repression domain, partially mediating its activity; binding of Cyp33 to the adjacent MLL-PHD domain potentiates this binding. Because the MLL repression domain activity was only partially relieved with the histone deacetylase inhibitor trichostatin A, we explored other protein interactions with this domain. Polycomb group proteins HPC2 and BMI-1 and the corepressor C-terminal-binding protein also bind the MLL repression domain. Expression of exogenous BMI-1 potentiates MLL repression domain activity. Functional antagonism between Mll and Bmi-1 has been shown genetically in murine knockout models for Mll and Bmi-1. Our new data suggest a model whereby recruitment of BMI-1 to the MLL protein may be able to modulate its function. Furthermore, repression mediated by histone deacetylases and that mediated by polycomb group proteins may act either independently or together for MLL function in vivo.

Role for Id-1 in immunobiology of normal keratinocytes and in basal cell carcinoma.

It has been established that Id proteins can block the basic helix-loop-helix (HLH) transcription factors, thereby impacting the onset of senescence in keratinocytes, as well as influencing tumorigenesis involving squamous cell carcinomas. However, the ability of Id-1 to influence the immunologic response of epithelial cells to cytokines implicated in cutaneous oncology such as gamma interferon (IFN-gamma) has not been determined. Using a whole population of human keratinocytes infected with a retrovirus to induce over-expression of Id-1, the influence on early differentiation of rapidly proliferating keratinocytes was assessed, as was the response to IFN-gamma. While induction of involucrin, a marker of early differentiation, was not altered in Id-1 overexpressing keratinocytes, the IFN-gamma mediated increase in intercellular adhesion molecule-1 (ICAM-1) and HLA-DR was reduced. No change in constitutive or inducible levels of MHC class I antigen, CD95 (Fas antigen) or LFA-3 (CD58) was observed in this system. Immunostaining and Western blot analysis revealed over-expression of Id-1 in basal cell carcinomas (BCCs). These tumors not only strongly and diffusely expressed Id-1, but were also characterized by reduced ICAM-1 and HLA-DR expression. Thus, dysregulated Id-1 may not only contribute to delaying the senescence program in keratinocytes, it may also contribute to the escape of the relatively undifferentiated tumor cells in BCC from immune surveillance.

Apoptotic stimuli initiate MLL-AF9 translocations that are transcribed in cells capable of division.

Activation of apoptosis introduces a site-specific break within intron 11 of the MLL gene. Using the CD95 apoptotic signaling pathway in human lymphoblastoid cells, the 5' fragment of MLL undergoes translocation to intron 4 of AF9 and the proleukemogenic MLL-AF9 fusion gene created is transcribed. Both the breaks in MLL and transcription of the MLL-AF9 fusion gene are suppressed in the presence of the broad spectrum caspase inhibitor, zVAD.fmk. Duplicate cells containing sequence identical MLL-AF9 fusion junctions were identified within a cell population that had recovered from apoptosis. This indicated that cells harboring a translocation initiated by apoptotic cleavage had divided. These data are consistent with a novel pathogenic role for the apoptotic program where translocations with leukemogenic potential are created within cells that have the capacity to divide.

Role of INK4a/Arf locus-encoded senescent checkpoints activated in normal and psoriatic keratinocytes.

During malignant transformation in skin, epidermal keratinocytes (KCs) frequently acquire the capacity to by-pass cellular senescence, a response that normally limits their unrestricted proliferation. Despite growing interest in the role for senescence during aging of skin and cutaneous carcinogenesis, little is known regarding regulation of three proteins encoded by the INK4a/ARF locus (p12, p14(ARF), p16) in KCs. In this study, several molecular pathways are explored using cultured KCs and KCs freshly isolated from psoriatic plaques. p16 and p14(ARF) are predominantly expressed spontaneously when foreskin-derived early-passage KCs undergo confluency-induced premature senescence. Induction of p14(ARF) on confluency occurred with low E2F-1 levels. Suspension of KCs in methylcellulose induced p12 expression. Addition of various cytokines (interferon-gamma, tumor necrosis factor-alpha) or a phorbol ester [12-O-tetradecanoylphorbol-13-acetate (TPA)] only induced p16, but not p14(ARF). Confluent KCs up-regulated Ras activity and the downstream signaling involving ERK. Addition of MAPK inhibitor blocked cytokine and TPA-induced p16 expression. Confluency and interferon-gamma induced premature senescence and p16 expression was linked to induction of the transcription factor Egr-1. KCs derived from chronic psoriatic plaques were characterized by enhanced p16, p14(ARF), and p12 expression accompanied by elevated Egr-1 levels. These results demonstrate that multiple and highly divergent stimuli can trigger the senescent checkpoint in human KCs with differential regulation of p16, p14(ARF), and p12. Although abnormal mitogenic signaling by oncogenic Ras is generally cited as being responsible for induction of premature senescence, our findings indicate that a broader perspective is warranted, to include confluency and cytokine-/TPA-induced pathways for KCs.

A new family of cyclophilins with an RNA recognition motif that interact with members of the trx/MLL protein family in Drosophila and human cells.

A new family of cyclophilins with an RNA recognition motif (RRM) has members in vertebrates, roundworms and flatworms. We have identified a Drosophilacyclophilin, Dcyp33, with a high degree of amino acid sequence identity and similarity with other members of the family. Dcyp33 interacts through its RRM domain with the third PHD finger of trithorax. This interaction is conserved in the human homologues of these proteins, Cyp33 and MLL. Over expression of Dcyp33 in DrosophilaSL1 cells results in down-regulation of AbdominalB Hoxgene expression, mirroring the effect of human Cyp33 on the expression of human HOXgenes.