Identification, Cloning, Expression, and Biochemical Characterization of the Testis-specific RNA Polymerase II Elongation Factor ELL3

Abstract

The human ELL gene, which is a frequent target for translocation in acute myeloid leukemia, was initially isolated from rat liver nuclei and found to be an RNA polymerase II elongation factor. Based on homology to ELL, we later cloned ELL2 and demonstrated that it can also increase the catalytic rate of transcription elongation by RNA polymerase II. To better understand the role of ELL proteins in the regulation of transcription by RNA polymerase II, we have initiated a search for proteins related to ELLs. In this report, we describe the molecular cloning, expression, and characterization of ELL3, a novel RNA polymerase II elongation factor approximately 50% similar to both ELL and ELL2. Our transcriptional studies have demonstrated that ELL3 can also increase the catalytic rate of transcription elongation by RNA polymerase II. The C-terminal domain of ELL, which we recently demonstrated to be required and sufficient for the immortalization of myeloid progenitor cells, shares strong similarities to the C-terminal domain of ELL3. ELL3 was localized by immunofluorescence to the nucleus of cells, and Northern analysis indicated that ELL3 is a testis-specific RNA polymerase II elongation factor.Many cellular factors involved in human oncogenesis have been identified as genes at breakpoints of frequently occurring chromosomal translocations. The protein products of some of these genes are transcriptional factors that regulate the general or specific expression of many genes. The ELL gene was initially identified on chromosome 19p13.1, which undergoes frequent translocation with the trithorax-like MLL (ALL-1, HRX) gene on chromosome 11q23 in acute myeloid leukemia (1, 2). ELL is a 621 amino acid-containing protein that can increase the catalytic rate of transcription elongation of RNA polymerase II by suppressing transient pausing at multiple sites along the DNA from both promoter-dependent and promoter-independent templates (3-5). To date, eight elongation factors have been defined biochemically (17,24). These factors are named SII (6 -9), P-TEFb (10 -11), DSIF (11-13), factor 2 (11, 14), TFIIF (15), elongin (SIII) (16), ELL (3, 18), and ELL2 (19). These RNA polymerase II elongation factors fall into several functional classes. Some can prevent arrest, like PTEFb and SII. Some can regulate the rate of transcription elongation through nucleosomes, such as FACT (20). Others operate to increase the catalytic rate of transcription elongation by altering the K m and/or the V max of the polymerase, such as TFIIF, elongin (SIII), the ELL complex, and ELL2 (4, 5).In an effort to better understand how transcription elongation by RNA polymerase II is controlled under normal conditions and in disease states, we are attempting to reconstitute RNA polymerase II transcription elongation machinery in vitro. In so doing, we have now identified and cloned a novel ELL family member, ELL3, and characterized its biochemical role in regulating transcription elongation. MATERIALS AND METHODS Cloning and Expression of ELL3-Searches of GenB...

dELL is an essential RNA polymerase II elongation factor with a general role in development

“…5B). Because both the ct 53d and N nd-1 mutations reduce gene expression (25,26), the dominant enhancement of these mutations by Su(Tpl) indicates that dELL normally increases expression of ct and N. Discussion ELL family of proteins have been characterized biochemically on the basis of their ability to promote Pol II elongation activity in vitro by suppressing transient pausing on a purified DNA template (8,10,13,14). This suggests an important role for ELL-dependent elongation in gene expression.…”

“…Each increases the catalytic rate of transcription elongation by RNA Pol II. ELL and ELL2 are expressed in most or all tissues (8,11,15), whereas ELL3 is expressed only in testis (14). Although ELL homologs are found in Caenorhabditis elegans, Drosophila, Xenopus, teleost fishes, and mammals, they are absent from both budding and fission yeast, suggesting that the ELL family of elongation factors represents an adaptation of metazoans.…”

“…Three mammalian ELL family proteins-ELL, ELL2, and ELL3-have been described (8,13,14). Each increases the catalytic rate of transcription elongation by RNA Pol II.…”

ELL-associated factors 1 and 2 are positive regulators of RNA polymerase II elongation factor ELL

“…ELL and ELL2 are expressed in a wide variety of tissues in humans. ELL3, on the other hand, appears to be expressed predominantly in the testis (5,6).…”

“…In addition to ELL, human cells have two ELL paralogs, ELL2 and ELL3, which can also stimulate the rate of elongation by pol II in vitro (5,6). ELL and ELL2 are expressed in a wide variety of tissues in humans.…”