Transcription elongation factor DSIF/Spt4-Spt5 is capable of promoting and inhibiting RNA polymerase II elongation and is involved in the expression of various genes. While it has been known for many years that DSIF inhibits elongation in collaboration with the negative elongation factor NELF, how DSIF promotes elongation is largely unknown. Here, an activity-based biochemical approach was taken to understand the mechanism of elongation activation by DSIF. We show that the Paf1 complex (Paf1C) and Tat-SF1, two factors implicated previously in elongation control, collaborate with DSIF to facilitate efficient elongation. In human cells, these factors are recruited to the FOS gene in a temporally coordinated manner and contribute to its high-level expression. We also show that elongation activation by these factors depends on P-TEFb-mediated phosphorylation of the Spt5 C-terminal region. A clear conclusion emerging from this study is that a set of elongation factors plays nonredundant, cooperative roles in elongation. This study also shows unambiguously that Paf1C, which is generally thought to have chromatin-related functions, is involve directlyd in elongation control.

. However, the physiological roles of this negative regulation are not well understood. Here, by using a number of approaches to identify protein-DNA interactions in vivo, we show that DSIF-and NELF-mediated transcriptional pausing has a dual function in regulating immediate-early expression of the human junB gene. Before induction by interleukin-6, RNAPII, DSIF, and NELF accumulate in the promoter-proximal region of junB, mainly at around position ؉50 from the transcription initiation site. After induction, the association of these proteins with the promoter-proximal region continues whereas RNAPII and DSIF are also found in the downstream regions. Depletion of a subunit of NELF by RNA interference enhances the junB mRNA level both before and after induction, indicating that DSIF-and NELF-mediated pausing contributes to the negative regulation of junB expression, not only by inducing RNAPII pausing before induction but also by attenuating transcription after induction. These regulatory mechanisms appear to be conserved in other immediate-early genes as well.

Recent estimates indicate that 60% of human genes include alternative polyadenylation sites. Hence, control of alternative polyadenylation can have a great impact on gene expression and cellular function. Cleavage factor (CF) Im is a 3¢-end processing factor that is essential for in vitro processing. CFIm purified from HeLa cells is associated with three polypeptides of 25, 59 and 68 kD, and it is generally thought to be a heterodimer composed of the 25-kD subunit and one of the larger subunits. Previously, we serendipitously discovered that knockdown of CFIm25 causes an upstream shift in the utilization of alternative polyadenylation sites. Here, we investigated whether this is because of an inherent property of the CFIm complex and, if so, what structural elements are important for its function. The major conclusions of this study are that (i) contrary to previous assumptions, CFIm forms stable heterotetramers through dimerization of CFIm25 and (ii) the CFIm complex per se is responsible for the control of alternative polyadenylation. (iii) However, the structurally related CFIm68 and CFIm59 are functionally redundant and (iv) CFIm68 appears to have a higher specific activity. Thus, this study establishes that CFIm not only plays a general role in 3¢-end processing but also plays a regulatory role in poly(A) site selection.

bRestores TBP function 1 (Rtf1) is generally considered to be a subunit of the Paf1 complex (PAF1C), a multifunctional protein complex involved in histone modification and transcriptional or posttranscriptional regulation. Rtf1, however, is not stably associated with the PAF1C in most species except Saccharomyces cerevisiae, and its biochemical functions are not well understood. Here, we show that human Rtf1 is a transcription elongation factor that may function independently of the PAF1C. Rtf1 requires "Rtf1 coactivator" activity, which is most likely unrelated to the PAF1C or DSIF, for transcriptional activation in vitro. A mutational study revealed that the Plus3 domain of human Rtf1 is critical for its coactivator-dependent function. Transcriptome sequencing (RNA-seq) and chromatin immunoprecipitation studies in HeLa cells showed that Rtf1 and the PAF1C play distinct roles in regulating the expression of a subset of genes. Moreover, contrary to the finding in S. cerevisiae, the PAF1C was apparently recruited to the genes examined in an Rtf1-independent manner. The present study establishes a role for human Rtf1 as a transcription elongation factor and highlights the similarities and differences between the S. cerevisiae and human Rtf1 proteins. Restores TBP (TATA box-binding protein) function 1 (Rtf1) was identified as a suppressor of a TBP mutant in Saccharomyces cerevisiae (1). Subsequent genetic and biochemical studies in yeast have shown that Rtf1 functions as a component of the polymerase-associated factor 1 (Paf1) complex (PAF1C) containing Paf1, Ctr9, Leo1, and Cdc73 (2-5). The PAF1C is a multifunctional protein complex whose primary role is to facilitate histone modifications, such as H2B monoubiquitination at K123 and H3 methylation at K4 and K79 (6-12). The PAF1C also plays important roles in transcription elongation through chromatin, as well as on naked DNA (13-16). Furthermore, the PAF1C is involved in transcription termination and 3= processing of polyadenylated and nonpolyadenylated .Paf1, Ctr9, Leo1, Cdc73, and Rtf1 are highly conserved in eukaryotes; however, the subunit compositions of the PAF1C vary among species. Purification of the PAF1C from human HeLa cells yielded a five-subunit complex lacking Rtf1 but containing Ski8 as an additional subunit (22,26,27). Similarly, coimmunoprecipitation studies in zebrafish, Drosophila, and Schizosaccharomyces pombe showed that PAF1C homologs in these species lack a stably associated Rtf1 subunit (28-30). At the functional level, however, Rtf1 and other PAF1C subunits have a number of similarities. For example, knockout or knockdown of Rtf1 and other PAF1C subunits results in similar defects in histone modifications in all the species examined (27,28,31). At the organismal level, inhibition of Rtf1 and other PAF1C subunits causes similar defects in epidermal morphogenesis in Caenorhabditis elegans and in somitogenesis and cardiomyocyte development in zebrafish (29,32,33). Rtf1 and Paf1 colocalize with each other and with RNA polymerase II (RNAPII) in...

Accession number. All the RNA-seq data generated in this study have been deposited in the Gene Expression Omnibus under accession number GSE73632.