The human ELL gene on chromosome 19 undergoes frequent translocation with the trithorax-like MLL gene on chromosome 11 in acute myeloid leukemia. Recently, it was demonstrated that the product of the human ELL gene encodes an RNA polymerase II elongation factor In addition to its elongation regulatory activity, ELL contains a novel type of RNA polymerase II interaction domain that is capable of negatively regulating polymerase activity in promoter-specific transcription in vitro (Shilatifard,

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... , Haque, D., Conaway, R. C., and Conaway, J. W. (1997) J. Biol. Chem. 272, 22355-22363). Here, we report the identification and purification of a large ELL-containing complex that contains three proteins in addition to ELL and that we have named the Holo-ELL complex. The Holo-ELL complex can increase the catalytic rate of transcription elongation by RNA polymerase II. However, unlike the ELL polypeptide alone, the Holo-ELL complex is not capable of negatively regulating polymerase activity in promoter-specific transcription in vitro. The inability of the Holo-ELL complex to negatively regulate polymerase activity in promoterspecific transcription suggests that one or more of the ELL-associated proteins regulate this activity, possibly through an interaction with the N-terminal domain of the ELL protein, which was shown to be required for the transcriptional inhibitory activity of ELL. Characterization of these ELL interacting proteins should help define the regulation of the biochemical activities of ELL and how loss of this regulation leads to the development of acute myeloid leukemia. The human ELL gene was initially identified as a gene that undergoes frequent translocations with the trithorax-like MLL gene in acute myeloid leukemia (AML) (1, 2). 1 We demonstrated that ELL, a basic 621-amino acid protein, interacts directly with Pol II (3, 4) and can increase the catalytic rate of transcription elongation by Pol II by suppressing transient pausing at multiple sites along the DNA (5). ELL can also regulate the transcription by a novel Pol II interaction domain that is capable of negatively regulating polymerase activity in promoter-specific transcription initiation in vitro (3). Remarkably, the MLL-ELL translocation, which is found in patients with AML, results in the deletion of a portion of this functional domain (1, 2). ELL mutants lacking the sequence that is deleted by the translocation are fully active in elongation and can interact with Pol II. However, such mutants fail to inhibit initiation by Pol II (3). It is not yet clear how the transcription initiation inhibitory activity of ELL normally plays a role in the regulation of gene expression or the regulation of the cell cycle. However, further studies on the biochemical mechanism and physiological function of this functional domain of ELL and proteins that interact with this domain will improve our understanding of the role of this domain of the ELL protein in transcriptional regulation and in development of leukemia. The original purification of ELL took advantage of hydrophobic interactions and reverse-phase chromatography to purify a single polypeptide that was active as an RNA polymerase II elongation factor (4). This purification employed denaturing solvents and conditions that destroy multiprotein complexes, and the proteins associated with ELL were not identified initially. However, alternate methods of purification procedures demonstrated that ELL exists as a multiprotein complex in vivo. As part of our effort to understand how elongation by RNA polymerase II is normally controlled and how it is dysregulated in certain malignancies, we set out to purify this ELL-containing complex. In this report, we describe the purification and characterization of a novel ELL-containing complex that we have named the Holo-ELL complex. EXPERIMENTAL PROCEDURES Materials-Ultrapure ribonucleoside 5Ј-triphosphates were purchased form Amersham Pharmacia Biotech. [␣-32 P]CTP was obtained from Andotech. Leupeptin, antipeptin, phenylmethylsulfonyl fluoride, and heparin were obtained from Sigma. Bovine serum albumin (Pentex fraction V) and Western development reagents were obtained from ICN ImmunoBiologicals. Glycerol (spectranalyzed grade), potassium chloride, Hepes, Tris, Ammonium Sulfate, and ultrapure sucrose were purchased from Fisher. The chromatographic columns DEAE-5PW and SP-5PW were purchased from Tosohaas, and Mono-Q, Mono-S, Suprose-12 HR, Suprose-6 PC, and Mini-Q were purchased from Amersham Pharmacia Biotech. Step I: Purification of the Holo-ELL Complex-Rat liver extract was prepared by homogenization of 180 rat livers as described previously (4), with the exception that nuclear and cytosolic extract were pooled together after the removal of the lysosomal fraction. The purification of the holo-ELL was performed at 4°C, and the fractions were not frozen unless indicated. Step II: DEAE-cellulose-The 0 -40% (NH 4 ) 2 SO 4 fraction was dialyzed to a conductivity equivalent to that of 100 mM (NH 4 ) 2 SO 4 in buffer A (40 mM Tris-HCl, pH 7.9, 10% glycerol, 1 mM EDTA, 1 mM DTT, 0.5 mM PMSF) and was then mixed with 1.0 liter of DEAE-cellulose equil-